Crystallization of M-CSFα

ABSTRACT

The present invention is directed to methods for crystallizing macrophage colony stimulating factor (M-CSF) and to a crystalline M-CSF produced thereby. The present invention is also directed to methods for designing and producing M-CSF agonists and antagonists using information derived from the crystallographic structure of M-CSF. The invention is also directed to methods for screening M-CSF agonists and antagonists. In addition, the present invention is directed to an isolated, purified, soluble and functional M-CSF receptor.

Work described herein was funded with Government support. The Governmenthas certain rights in inventions arising as part of that work.

This application is a continuation-in-part of U.S. Ser. No. 07/896,512filed Jun. 9, 1992, now abandoned, from which priorities are claimedunder 35 USC §120.

FIELD OF THE INVENTION

The present invention relates in general to crystalline compositions ofmacrophage colony stimulating factor "M-CSF" and in particular tomethods for the use of structural information (including X-raydiffraction patterns) of crystalline M-CSF for agonist and antagonistproduction, as well as assays for detection of same.

BACKGROUND OF THE INVENTION

Monocyte-macrophage colony-stimulating factor is produced by a varietyof cells, including macrophages, endothelial cells and fibroblasts (see,Ralph et al., "The Molecular and Biological Properties of the Human andMurine Members of the CSF-1 Family" in Molecular Basis of LymphokineAction, Humana Press, Inc., (1987), which is incorporated herein byreference). M-CSF is composed of two "monomer" polypeptides, which forma biologically active dimeric M-CSF protein (hereinafter referred to as"M-CSF dimer"). M-CSF belongs to a group of biological agonists thatpromote the production of blood cells. Specifically, it acts as a growthand differentiation factor for bone marrow progenitor cells of themononuclear phagocyte lineage. Further, M-CSF stimulates theproliferation and function of mature macrophages via specific receptorson responding cells. In clinical trials M-CSF has shown promise as apharmaceutical agent in the correction of blood cell deficienciesarising as a side-effect of chemotherapy or radiation therapy for cancerand may be beneficial in treating fungal infections associated with bonemarrow transplants. M-CSF may also play significant biological roles inpregnancy, uveitis, and atherosclerosis. Development of M-CSF agonistsor antagonists may prove to be of value in modifying the biologicalevents involved in these conditions.

M-CSF exists in at least three mature forms: short (M-CSFα),intermediate (M-CSFγ), and long (M-CSFβ). Mature M-CSF is defined asincluding polypeptide sequences contained within secreted M-CSFfollowing amino terminus processing to remove leader sequences andcarboxyl terminus processing to remove domains including a putativetransmembrane region. The variations in the three mature forms are dueto alternative mRNA splicing (see, Cerretti et al. Molecular Immunology,25:761 (1988)). The three forms of M-CSF are translated from differentmRNA precursors, which encode polypeptide monomers of 256 to 554 aminoacids, having a 32 amino acid signal sequence at the amino terminal anda putative transmembrane region of approximately 23 amino acids near thecarboxyl terminal. The precursor peptides are subsequently processed byamino terminal and carboxyl terminal proteolytic cleavages to releasemature M-CSF. Residues 1-149 of all three mature forms of M-CSF areidentical and are believed to contain sequences essential for biologicalactivity of M-CSF. In vivo M-CSF monomers are dimerized viadisulfide-linkage and are glycosylated. Some, if not all, forms of M-CSFcan be recovered in membrane-associated form. Such membrane-bound M-CSFmay be cleaved to release secreted M-CSF. Membrane associated M-CSF isbelieved to have biological activity similar to M-CSF, but may haveother activities including cell-cell association or activation.

Polypeptides, including the M-CSFs, have a three-dimensional structuredetermined by the primary amino acid sequence and the environmentsurrounding the polypeptide. This three-dimensional structureestablishes the polypeptide's activity, stability, binding affinity,binding specificity, and other biochemical attributes. Thus, a knowledgeof a protein's three-dimensional structure can provide much guidance indesigning agents that mimic, inhibit, or improve its biological activityin soluble or membrane bound forms.

The three-dimensional structure of a polypeptide may be determined in anumber of ways. Many of the most precise methods employ X-raycrystallography (for a general review, see, Van Holde, PhysicalBiochemistry, Prentice-Hall, N. J. pp. 221-239, (1971), which isincorporated herein by reference). This technique relies on the abilityof crystalline lattices to diffract X-rays or other forms of radiation.Diffraction experiments suitable for determining the three-dimensionalstructure of macromolecules typically require high-quality crystals.Unfortunately, such crystals have been unavailable for M-CSF as well asmany other proteins of interest. Thus, high-quality, diffractingcrystals of M-CSF would assist the determination of itsthree-dimensional structure.

Various methods for preparing crystalline proteins and polypeptides areknown in the art (see, for example, McPherson, et al. "Preparation andAnalysis of Protein Crystals", A. McPherson, Robert E. KriegerPublishing Company, Malabar, Fla. (1989); Weber, Advances in ProteinChemistry 41:1-36 (1991); U.S. Pat. No. 4,672,108; and U.S. Pat. No.4,833,233; all of which are incorporated herein by reference for allpurposes). Although there are multiple approaches to crystallizingpolypeptides, no single set of conditions provides a reasonableexpectation of success, especially when the crystals must be suitablefor X-ray diffraction studies. Thus, in spite of significant research,many proteins remain uncrystallized.

In addition to providing structural information, crystallinepolypeptides provide other advantages. For example, the crystallizationprocess itself further purifies the polypeptide, and satisfies one ofthe classical criteria for homogeneity. In fact, crystallizationfrequently provides unparalleled purification quality, removingimpurities that are not removed by other purification methods such asHPLC, dialysis, conventional column chromatography, etc. Moreover,crystalline polypeptides are often stable at ambient temperatures andfree of protease contamination and other degradation associated withsolution storage. Crystalline polypeptides may also be useful aspharmaceutical preparations. Finally, crystallization techniques ingeneral are largely free of problems such as denaturation associatedwith other stabilization methods (e.g. lyophilization). Thus, thereexists a significant need for preparing M-CSF compositions incrystalline form and determining their three-dimensional structure. Thepresent invention fulfills this and other needs. Once crystallizationhas been accomplished, crystallographic data provides useful structuralinformation which may assist the design of peptides that may serve asagonists or antagonists. In addition, the crystal structure providesinformation useful to map, the receptor binding domain which could thenbe mimicked by a small non-peptide molecule which may serve as anantagonist or agonist.

SUMMARY OF THE INVENTION

The present invention provides crystalline forms of M-CSF dimers.Preferably, the dimers are formed from polypeptides containing between146 to 162 amino acids residues at or near the N-terminus of matureM-CSF (e.g. glu₁ glu₂ val₃ . . . ). In a specific embodiment, thepolypeptide includes residues 4 to 158 of mature M-CSFα polypeptide,preferably in the non-glycosylated form.

Another aspect of the invention provides a method of crystallizing anM-CSF. A preferred crystallization method according to the presentinvention includes the following steps: mixing a preselected,substantially pure M-CSF dimer and a precipitant to form an M-CSFmixture; precipitating crystalline M-CSF from the mixture; and isolatingthe M-CSF in crystalline form. In some specific embodiments, theprecipitant contains polyethylene glycol. Other components such asammonium sulfate and/or other ionic compounds may be added to thesolution. It has been found by x-ray crystallography that M-CSF producedby the method of the present invention can crystallize into the P2₁ 2₁2₁ space groups for example.

Variations of the crystallization method are also provided. For example,the step of precipitating crystals from the M-CSF mixture may involveequilibrating the M-CSF mixture with a second mixture. The secondmixture is typically a solution that consists of a higher concentrationof precipitant than the first M-CSF mixture. The step of equilibratingpreferably consists of applying the M-CSF mixture to a surface andallowing the applied M-CSF mixture to come into equilibrium with areservoir of the second mixture. In other embodiments, the step ofprecipitating M-CSF crystals is initiated by seeding the M-CSF mixturewith seed crystals or altering the temperature of the M-CSF mixture.Another aspect of the invention involves identifying compounds that havestructures that mimic a receptor binding region of the three-dimensionalstructure of M-CSF to varying degrees and can in many instances functionas M-CSF agonists or antagonists. Compounds that interact with thereceptor-binding region of M-CSF may be antagonists. Thethree-dimensional alpha-carbon coordinates of a truncated M-CSF dimer ispresented in Appendix 1. In one embodiment of the present invention, thethree-dimensional structure of M-CSF is obtained by first crystallizingan M-CSF dimer (having M-CSF receptor-binding residues) to form at leastone M-CSF crystal. Next, a source of radiation is used for irradiatingan M-CSF crystal to obtain a diffraction pattern of the M-CSF crystal.Finally, a three-dimensional structure of M-CSF is obtained from thediffraction pattern. In most embodiments, the three-dimensionalstructure includes an M-CSF receptor-binding region.

The present invention is also directed to a method for selectingcandidate amino acid substitutions in a protein, based on structuralinformation, and more particularly M-CSF, comprising determining thethree-dimensional structure of M-CSF by the methods of the presentinvention; followed by determining the solvent accessible amino acidresidues of the protein, determining which residues are not involved indimer formation. Applying these criteria, amino acids in M-CSF which aresolvent accessible and which are not involved in dimer formation areselected for substitution with non-conservative amino acids. SinceM-CSFβ has intrachain disulfide bonds involving cysteines 157 and/or159, we believe the C-terminal region of M-CSF extends from the "rear"of the structure we have solved, providing a variable-length "tether"for membrane-bound forms of M-CSF. Thus, the "front" or receptor-bindingregion of M-CSF is on the opposite side of the molecules, consisting ofsolvent-accessible residues in or near helices A, C, and D, includingresidues from about 6 to 26, 71 to 90, and 110 to 130, respectively, ofnative M-CSF. Preferred amino acids for substitution and preferredsubstituting amino acids include but are not limited to: H15→A or L;Q79→A or D; R86→E or D; E115→A; E41→K or R; K93→A or E; D99→K or R;L55→Q or N; S18→A or K; Q20→A or D; I75→K or E; V78→K or R; L85→E or N;D69→K or R; N70→A or E; H9→A or D; N63→K or R; and T34→Q or K. Mostpreferred are those substitutions give rise to novel M-CSF agonists andM-CSF antagonists. Additionally, the present invention is also directedto a method for producing antagonists and agonists by substituting atleast one and preferably fewer than 5 solvent accessible residues perM-CSF monomer.

The invention is also directed to heterodimeric M-CSF in which only onesubunit contains substituted solvent accessible amino acids involved insignal transduction and to heterodimeric M-CSF in which each subunitcontains different substituted solvent accessible amino acids involvedin signal transduction. The present invention is also directed to M-CSFhaving amino acid substitutions which do not impair binding to the M-CSFreceptor. Screening for agonists and antagonists is then accomplishedusing bioassays and receptor binding assays using methods well known inthe art, including those described in the Examples below.

In addition the invention is directed to an isolated, purified, solubleand functional M-CSF receptor. The present invention is also directed toa method for screening M-CSF agonists and antagonists using a solubleM-CSF receptor.

A further understanding of the present invention can be obtained byreference to the drawings and discussion of specific embodiments.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a section of a diffraction pattern of an M-CSFα crystalprepared according to the present invention;

FIG. 2 is a topology diagram showing the disulfide bonds in truncateddimeric M-CSF;

FIG. 3 is a stereodiagram of the C-alpha backbone with every tenthresidue labelled and with the non-crystallographic symmetry axisindicated by a dotted line;

FIGS. 4A and 4B present two views of a ribbon diagram highlighting thesecondary structural elements of M-CSF. In FIGS. 4A and 4B cysteineresidues have been represented by a ball-and-stick model and in FIG. 4Bthe non-crystallographic symmetry axis is indicated by a dotted line;

FIGS. 5A-5C illustrate size-exclusion HPLC analysis of the NΔ3CΔ158M-CSF short clone homodimer (158)(FIG. 5A), NΔ3CΔ221 C157S, C159S longclone homodimer (221F, FIG. 5B)(FIG. 5B), and the short clone/long cloneheterodimer (158/221F, FIG. 5C)(FIG. 5C) and their correspondingbiological activities;

FIGS. 6A-6C illustrates size exclusion HPLC(A) and both non-reduced(B)and reduced(C) SDS-PAGE analysis of the preparative purification ofM-CSF; more particularly, FIG. 6 graphically illustrates the separationon Phenyl-HPLC size exclusion chromatography of the three species ofM-CSF dimers of FIGS. 5A-5C, i.e., the 158 homodimer, the 221F homodimerand the 158/221F heterodimer, and indicates that the three absorbancepeaks at 280 nm (solid line) correlate with M-CSF activity in U/ml×10⁻⁶(dotted line); FIG. 6 illustrates an SDS-PAGE analysis undernon-reducing conditions of the preparative purification of the 158/221Fheterodimer (intermediate molecular weight species) relative to the 158homodimer (lower molecular weight species) and the 221F homodimer(highest molecular weight species); FIG. 6 illustrates an SDS-PAGEanalysis under reducing conditions of the preparative purification ofthe 158/221F heterodimer (middle lanes) relative to the 158 homodimer(left lanes) and the 221F homodimer (right lanes); and

FIG. 7 illustrates the competitive binding of M-CSF and M-CSF muteins toNFS60 cell M-CSF receptors. In FIG. 7, competitive binding curves areshown for M-CSFαNΔ3CΔ158 (closed circles); M-CSFαNΔ3CΔ158 H9A,H15A/M-CSFβNΔ3CΔ221 C157S, C159S heterodimer (closed squares); dimericQ20A, V78KF mutein (open circles); and dimeric H9A, H15A mutein (opensquares).

DESCRIPTION OF THE PREFERRED EMBODIMENTS DEFINITIONS

As used herein "M-CSF polypeptide" refers to a human polypeptide havingsubstantially the same amino acid sequence as the mature M-CSFα, M-CSFβ,or M-CSFγ polypeptides described in Kawasaki et al., Science. 230:291(1985), Cerretti et al., Molecular Immunology, 25:761 (1988), or Ladneret al., EMBO Journal6:2693 (1987), each of which are incorporated hereinby reference. Such terminology reflects the understanding that the threemature M-CSFs have different amino acid sequences, as described above.

Certain modifications to the primary sequence of M-CSF can be made bydeletion, addition, or alteration of the amino acids encoded by the DNAsequence without destroying the desired structure (e.g., the receptorbinding ability of M-CSF) in accordance with well-known recombinant DNAtechniques. Further, a skilled artisan will appreciate that individualamino acids may be substituted or modified by oxidation, reduction orother derivitization, and the polypeptide may be cleaved to obtainfragments that retain the active binding site and structuralinformation. Such substitutions and alterations result in polypeptideshaving an amino acid sequence which falls within the definition ofpolypeptide "having substantially the same amino acid sequence as themature M-CSFα, M-CSFβ, and M-CSFγ polypeptides."

For purposes of crystallization, preferred lengths of the M-CSFα, β or γmonomers are between about 145 and 180 amino acids (counting from themature amino termrinus), and more preferably between about 145 and 162amino acids long. A specific monomer that may be present in acrystallizable dimer is M-CSFα and is NΔ3M-CSFαCΔ158 (3 amino acids aredeleted from the amino terminus and the total length is 155 aminoacids). All lengths are inclusive. As used herein the term "M-CSFα(4-158)" denotes an M-CSF having amino acid residues 4 to 158 of themature, processed M-CSFα polypeptide. Other nomenclature designationsfor C-terminal and N-terminal truncations of native M-CSF are set forthin U.S. Pat. No. 4,929,700 which is incorporated herein by reference.

Crystallizable glycosylation variants of the M-CSF polypeptides areincluded within the scope of this invention. These variants includepolypeptides completely lacking in glycosylation and variants having atleast one fewer glycosylated site than the mature forms, as well asvariants in which the glycosylation pattern has been changed from thenative forms. Also included are deglycosylated and unglycosylated aminoacid sequence variants, as well as deglycosylated and unglycosylatedM-CSF subunits having the mature amino acid sequence (see, U.S. Pat. No.5,032,626).

"M-CSF" dimer refers to two M-CSF polypeptide monomers that havedimerized. M-CSF dimers may include two identical polypeptide monomers(homodimers) or two different polypeptide monomers (heterodimers such asan M-CSFα-M-CSFβ dimer, an M-CSF long clone and short clone dimer).M-CSF monomers may be converted to M-CSF dimers in vitro as described inU.S. Pat. No. 4,929,700, which is incorporated herein by reference.Recombinantly expressed M-CSFs may also be variably glycosylated as theyexist in vivo, partially glycosylated, or completely lacking inglycosylation (unglycosylated). Glycosylated M-CSFs may be produced invivo with carbohydrate chains which may later be enzymaticallydeglycosylated in vitro.

Biologically active M-CSF exhibits a spectrum of activity understood inthe art. For instance, M-CSF stimulates the proliferation and functionof mature macrophages via specific receptors on responding cells.Further, M-CSF acts as a mononuclear phagocyte progenitor growth factor.The standard in vitro colony stimulating assay of Metcalf, J. CellPhysiol. 76:89 (1970) (which is incorporated herein by reference)results primarily in the formation of macrophage colonies when M-CSF isapplied to stem cells. Other biological assays are based on M-CSFinduced proliferation of M-CSF dependent cells such as the NFS-60 cellline. As used herein "M-CSF having biological activity" refers to M-CSF,including fragments and sequence variants thereof as described herein;that exhibit an art-recognized spectrum of activity with respect tobiological systems. Such M-CSF having biological activity will typicallyhave certain structural attributes in common with those of the matureM-CSF forms such as receptor binding site tertiary structure.

Agonists are substances that exhibit greater activity per se than thenative ligand while antagonists are substances that suppress, inhibit,or interfere with the biological activity of a native ligand. Agonistsand antagonists may be produced by the methods of the present inventionfor use in the treatment of diseases in which M-CSF has been implicatedeither as a potential treatment (e.g., for treating blood celldeficiencies arising as a side effect of chemotherapy treating fungalinfection associated with bone marrow transplants and others) or ashaving a role in the pathogenesis of the disease (e.g., ovarian cancer,uveitis, atherosclerosis).

Crystallization of M-CSF species in accordance with the presentinvention includes four general steps: expression, purification,crystallization and isolation.

Expression of Recombinant M-CSF

M-CSF crystallization requires an abundant source of M-CSF that may beisolated in a relatively homogeneous form. A variety of expressionsystems and hosts are suitable for the expression of M-CSF and will bereadily apparent to one of skill in the art. Because of the variabilityof glycosylation and other post-transnational modifications present inM-CSF produced in certain eukaryotic hosts, expression in E. coli mayprovide M-CSF with advantageous properties with regard tocrystallization. Typical in vitro M-CSF expression systems are describedin U.S. Pat. No. 4,929,700, for example.

For use in the present invention, a variety of M-CSF polypeptides canalso be readily designed and manufactured utilizing recombinant DNAtechniques well known to those skilled in the art. For example, theM-CSF amino acid sequence can vary from the naturally occurring sequenceat the primary structure level by amino acid substitutions, insertions,deletions, and the like. These modifications can be used in a number ofcombinations to produce the final modified polypeptide chain. Thepresent invention is useful for crystallizing such polypeptides anddimers thereof.

In general, modifications of the genes encoding the M-CSF polypeptideare readily accomplished by a variety of well-known techniques, such assite-directed mutagenesis (see, Gillman and Smith, Gene 8:81-97 (1979)and Roberts, S. et al., Nature 328:731-734 (1987) and U.S. Pat. No.5,032,676, all of which are incorporated herein by reference). Mostmodifications are evaluated by screening in a suitable assay for thedesired characteristic. For instance, a change in the M-CSFreceptor-binding character of the polypeptide can be detected bycompetitive assays with an appropriate reference polypeptides or by thebioassays described in U.S. Pat. No. 4,847,201, which is incorporatedherein by reference.

Insertional variants of the present invention are those in which one ormore amino acid residues are introduced into a predetermined site in theM-CSF. For instance, insertional variants can be fusions of heterologousproteins or polypeptides to the amino or carboxyl terminus of thesubunits. Substitutional variants are those in which at least oneresidue has been removed and a different residue inserted in its place.Nonnatural amino acids (i.e., amino acids not normally found in nativeproteins), as well as isosteric analogs (amino acid or otherwise) arealso suitable for use in this invention. Examples of suitablesubstitutions are well known in the art, such as the Glu→Asp, Ser→Cys,and Cys→Ser, His→alanine for example. Another class of variants aredeletional variants, which are characterized by the removal of one ormore amino acid residues from the M-CSF.

Other variants of the present invention may be produced by chemicallymodifying amino acids of the native protein (e.g., diethylpyrocarbonatetreatment which modifies histidine residues). Preferred or chemicalmodifications which are specific for certain amino acid side chains.Specificity may also be achieved by blocking other side chains withantibodies directed to the side chains to be protected. Chemicalmodification includes such reactions as oxidation, reduction, amidation,deamidation, or substitution of bulky groups such as polysaccharides orpolyethylene glycol (see e.g., U.S. Pat. No. 4,179,337 and WO91/21029both of which are incorporated herein by reference).

Exemplary modifications include the modification of lysinyl and aminoterminal residues by reaction with succinic or other carboxylic acidanhydrides. Modification with these agents has the effect of reversingthe charge of the lysinyl residues. Other suitable reagents formodifying amino-containing residues include imidoesters such as methylpicolinimidate; pyridoxal phosphate; pyridoxal chloroborohydride;trinitrobenzenesulfonic acid; O-methylisourea, 2,4-pentanedione; andtransaminaseN: talyzed reaction with glyoxylate, andN-hydroxysuccinamide esters of polyethylenene glycol or other bulkysubstitutions.

Arginyl residues may be modified by reaction with a number of reagents,including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, andninhydrin. Modification of arginine residues requires that the reactionbe performed in alkaline conditions because of the high pK_(a) of theguanidine functional group. Furthermore, these reagents may react withthe groups of lysine as well as the arginine epsilon-amino group.

Tyrosyl residues may also be modified with particular interest inintroducing spectral labels into tyrosyl residues by reaction witharomatic diazonium compounds or tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues may also be iodinated using ¹²⁵ I or ¹³¹ I to prepare labeledproteins for use in radioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) may be selectively modifiedby reaction with carbodiimides (R--N═C═N--R¹), where R and R¹ aredifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Conversely, glutaminyl and asparaginyl residues may be deamidated to thecorresponding glutamyl and aspartyl residues, respectively, under mildlyacidic conditions. Either form of these residues falls within the scopeof this invention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 1983!),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

The availability of a DNA sequence encoding M-CSF permits the use ofvarious expression systems to produce the desired polypeptides.Construction of expression vectors and recombinant production from theappropriate DNA sequences are performed by methods well known in theart. These techniques and various other techniques are generallyperformed according to Sambrook et al., Molecular Cloning --A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989),and Kriegler, M., Gene Transfer and Expression, A Laboratory Manual,Stockton Press, New York (1990), both of which are incorporated hereinby reference.

Purification of M-CSF

Purification steps are employed to ensure that the M-CSF is isolated,prior to crystallization, in a relatively homogeneous state. In general,a higher purity solution increases the likelihood of success ofsubsequent crystallization steps. Typical purification methods includethe use of centrifugation, partial fractionation using salt or organiccompounds, dialysis, conventional column chromatography (such asion-exchange, molecular sizing chromatography etc.), high performanceliquid chromatography (HPLC), and gel electrophoresis methods (see,e.g., Deutcher, "Guide to Protein Purification" in Methods in Enzymology(1990), Academic Press, Berkely, Calif., which is incorporated herein byreference for all purposes). Preferred purification conditions forgenerating unusually homogeneous M-CSF species as well as purificationof these species are disclosed, for example, in U.S. Pat. No. 4,929,700which is incorporated herein by reference. Other purification methodsare known and will be apparent to one of skill in the art.

Crystallization of M-CSF

Although many of the same physical principles govern crystallization ofpolypeptides (including M-CSF dimers) and small molecules, the actualcrystallization mechanisms differ significantly. For example, thelattice of small-molecule crystals effectively excludes solvent whilethat of polypeptide crystals includes substantial numbers of solventmolecules. Thus, special techniques must typically be applied tocrystallize polypeptides.

Polypeptide crystallization occurs in solutions where the polypeptideconcentration exceeds its solubility maximum (i.e., the polypeptidesolution is supersaturated). Such "thermodynamically metastable"solutions may be restored to equilibrium by reducing the polypeptideconcentration, preferably through precipitation of the polypeptidecrystals. Often polypeptides may be induced to crystallize fromsupersaturated solutions by adding agents that alter the polypeptidesurface charges or perturb the interactions between the polypeptide andbulk water to promote associations that lead to crystallization.

Compounds known as "precipitants" are often used to decrease thesolubility of the polypeptide in a concentrated solution. Precipitantsinduce crystallization by forming an energetically unfavorableprecipitant depleted layer around the polypeptide molecules. To minimizethe relative amount of this depletion layer, the polypeptides formassociations and ultimately crystals as explained in Weber, Advances inProtein Chemistry 41:1-36 (1991) which was previously incorporated byreferenceN, In addition to precipitants, other materials are sometimesadded to the polypeptide crystallization solution. These include buffersto adjust the pH of the solution (and hence surface charge on thepeptide) and salts to reduce the solubility of the polypeptide. Variousprecipitants are known in the art and include the following: ethanol,3-ethyl-2,4 pentanediol; and many of the polyglycols, such aspolyethylene glycol. A suitable precipitant for crystallizing M-CSF ispolyethylene glycol (PEG), which combines some of the characteristics ofthe salts and other organic precipitants (see, for example, Ward et al.,J. Mol. Biol. 98:161 1975! which is incorporated herein by reference forall purposes and McPherson J. Biol. Chem. 251:6300 1976!, which waspreviously incorporated by reference).

Commonly used polypeptide crystallization methods include the followingtechniques: batch, hanging drop, seed initiation, and dialysis. In eachof these methods, it is important to promote continued crystallizationafter nucleation by maintaining a supersaturated solution. In the batchniethod, polypeptide is mixed with precipitants to achievesupersaturation, the vessel is sealed and set aside until crystalsappear. In the dialysis method, polypeptide is retained in a sealeddialysis membrane which is placed into a solution containingprecipitant. Equilibration across the membrane increases the polypeptideand precipitant concentrations thereby causing the polypeptide to reachsupersaturation levels.

In the hanging drop technique, an initial polypeptide mixture is createdby adding a precipitant to concentrated polypeptide solution. Theconcentrations of the polypeptide and precipitants are such that in thisinitial form, the polypeptide does not crystallize. A small drop of thismixture is placed on a glass slide which is inverted and suspended overa reservoir of a second solution. The system is then sealed. Typicallythe second solution contains a higher concentration of precipitant orother dehydrating agent. The difference in the precipitantconcentrations causes the protein solution to have a higher vaporpressure than the solution. Since the system containing the twosolutions is sealed, an equilibrium is established, and water from thepolypeptide mixture transfers to the second solution. This equilibrationincreases the polypeptide and precipitant concentration in thepolypeptide solution. At the critical concentration of polypeptide andprecipitant, a crystal of the polypeptide will form. The hanging dropmethod is well known in the art (see, McPherson J. Biol. Chem. 251:63001976!, which was previously incorporated herein by reference).

Another method of crystallization introduces a nucleation site into aconcentrated polypeptide solution. Generally, a concentrated polypeptidesolution is prepared and a seed crystal of the polypeptide is introducedinto this solution. If the concentrations of the polypeptide and anyprecipitants are correct, the seed crystal will provide a nucleationsite around which a larger crystal forms.

In preferred embodiments, the crystals of the present invention will beformed from a dimer of M-CSF polypeptides. Preferred crystals aretypically at least about 0.2×0.2×0.05 mm, more preferably larger than0.4×0.4×0.4 mm, and most preferably larger than 0.5×0.5×0.5 mm. Aftercrystallization, the protein may be separated from the crystallizationmixture by standard techniques.

The crystals so produced have a wide range of uses. For example, highquality crystals are suitable for X-ray or neutron diffraction analysisto determine the three-dimensional structure of the M-CSF and, inparticular, to assist in the identification of its receptor bindingsite. Knowledge of the binding site region and solvent-accessibleresidues available for contact with the M-CSF receptor allows rationaldesign and construction of agonists and antagonist for M-CSFs.Crystallization and structural determination of M-CSF muteins havingaltered receptor binding ability or bioactivity allows the evaluation ofwhether such changes are caused by general structural deformation or byside chain alteration at the substitution site.

In addition, crystallization itself can be used as purification method.In some instances, a polypeptide or protein will crystallize from aheterogeneous mixture into crystals. Isolation of such crystals byfiltration, centrifugation, etc. followed by redissolving thepolypeptide affords a purified solution suitable for use in growing thehigh-quality crystals necessary for diffraction studies. Thesehigh-quality crystals may also be dissolved in water and then formulatedto provide an aqueous M-CSF solution having various uses known in theart including pharmaceutical purposes.

Of course, amino acid sequence variants of M-CSF may also becrystallized and used. These mutants can be used for, among otherpurposes, obtaining structural information useful for directingmodification of the binding affinity for M-CSF receptors. As with thenaturally occurring forms, the modified M-CSF forms may be useful aspharmaceutical agents for stimulating bone marrow proliferation,overcoming immune suppression and fungal diseases induced bychemotherapy, improving therapeutic efficacy, and lessening the severityor occurrence of side effects during therapeutic use of the presentinvention.

Furthermore, modified M-CSFs may be useful for treatment of disease inwhich soluble or membrane-bound M-CSF causes or exacerbates the diseasestate.

Characterization of M-CSF

After purification, crystallization and isolation, the subject crystalsmay be analyzed by techniques known in the art. Typical analysis yieldstructural, physical, and mechanistic information about the peptides. Asdiscussed above, X-ray crystallography provides detailed structuralinformation which may be used in conjunction with widely availablemolecular modeling programs to arrive at the three-dimensionalarrangement of atoms in the peptide. Exemplary modeling programs include"Homology" by Biosyrn (San Diego, Calif.), "Biograf" by BioDesign,"Nemesis" by Oxford Molecular, "SYBYL" and "Composer" by TriposAssociates, "CHARM" by Polygen (Waltham, Mass.), "AMBER" by Universityof California, San Francisco, and "MM2" and "MMP2" by Molecular Design,Ltd.

Peptide modeling can be used to design a variety of agents capable ofmodifying the activity of the subject peptide. For example, using thethree-dimensional structure of the active site, agonists and antagonistshaving complementary structures can be designed to enhance thetherapeutic utility of M-CSF treatment or to block the biologicalactivity of M-CSF. Further, M-CSF structural information is useful fordirecting design of proteinaceous or non-proteinaceous M-CSF agonistsand antagonists, based on knowledge of the contact residues between theM-CSF ligand and its receptor. Such residues are identified by the M-CSFcrystal structure as those which are solvent-accessible, distal to thecarboxyl terminal membrane anchoring region not involved in dimerinterface stabilizations, and possibly including residues not conservedbetween human and mouse M-CSF (which does not recognize the human M-CSFreceptor).

EXAMPLE 1

Systematic crystallization trials with M-CSF were made using the hangingdrop technique. A microdroplet (5μl) of mother liquor is suspended fromthe underside of a microscope cover slip, which is placed over a wellcontaining 1 ml of the precipitating solution. 60-70 initial trials wereset up, in which pH, temperature, counterion and precipitant werevaried. From these trials, the few that gave promising microcrystalswere picked for more careful examination.

It was discovered that suitable crystals may be grown from a 20 μl dropcontaining: 10 mg/ml protein, 100 mM MgCl₂, 50 mM Tris.Cl, pH 8.5, and12% PEG 4000. This drop was equilibrated against a reservoir containing24% PEG 4000. Tiny, needle-like crystals appeared in 2-3 days which wereredissolved in 10 μl water and recrystallized at room temperature. Goodquality chunky crystals appeared in 7-9 days in sizes ranging from0.3×0.3×0.3 mm to 0.5×0.5×1.0 mm.

Precession photographs revealed the space group to be P2₁ 2₁ 2₁ withunit cell dimensions: a=33.53 Å, b=65.11 Å, c=159.77 Å. This gives aunit cell volume of 349084.5 Å³, which is consistent with a dimer in thecrystallographic asymmetric unit, and 52% of the unit cell volume beingoccupied by solvent. The crystals diffracted to a resolution of 3 Å on aRigaku rotating anode X-ray generator (Danvers, Mass.) operated at 50 kVand 60 mA, and to 2.6 Å in synchrotron radiation.

Screening for heavy atom derivatives was done by soaking crystals intosolutions of heavy-metal salts. Zero-level precession pictures of thesoaks were used to identify potential derivatives. About 30 differentsoaking conditions were examined, of which 4 potential derivatives wereidentified. Unfortunately, some soaks caused the crystals to exhibitnon-isomorphism (i.e., the heavy atom soaks induced a change in celldimensions, making them unusable for phase calculation).

Three-dimensional intensity data were collected on film using anoscillation camera on the X-ray beam-line at the National SynchrotronLight Source, Brookhaven. Several other data sets, of native(underivatized M-CSF) as well as potential derivative crystals have beencollected on a Rigaku X-ray generator. The following data sets werecollected.

    ______________________________________              Resolution                       N          N    Crystal   (Å)  (observations)                                  (unique)                                        X-Ray Source    ______________________________________    Native    2.8      27922      7311  Synchrontron    Native    2.9      35236      7002  Rigaku                                        (film)    Native    3.5       5144      5116  Rigaku                                        (diffractometer)    K.sub.2 Hg(SCN).sub.4              3.5      15885      4119  Rigaku                                        (film)    UO.sub.2 Cl.sub.2              3.5      25492      5048  Rigaku                                        (film)    Cis-Pd(NH.sub.3).sub.2 Cl.sub.2              3.1      26122      6304  Synchrotron    ______________________________________

EXAMPLE 2

Recombinant M-CSF polypeptides were purified from E. coli. and renaturedto form a disulfide-linked dimeric protein as described in U.S. Pat. No.4,929,700. Crystallization of the resulting unglycosylated M-CSFαprotein (amino acids 4-158 in homodimeric form) was performed by thehanging drop method. Glass microscope plates were siliconized prior touse by dipping immersion into a 1% (volume:volume) solution of theorganosilane compound, Prosil-28 (PCR Incorporated, Gainesville, Fla.,32602) washing the treated glass plates with water, and baking at 180degrees.

A 2 mg/ml aqueous solution of purified human recombinant M-CSF wasdialyzed and concentrated against 50 mM Tris-HCl (pH 8.5) using adialysis tubing having a 10 kD cutoff. The final concentration ofpolypeptide (10 mg/ml) was determined by ultraviolet spectrophotometryat 280 nm.

About 7 microliters of the concentrated solution was mixed in each wellof the spot plate with 7 microliter of 20% (v/v) PEG 4000, 0.2M MgCl₂,0.1M Tris-HCl (pH 8.5). The spot plate was then placed in a clearplastic sandwich box containing 20 ml of 23% PEG 4000, 0.2M MgCl₂, 0.1MTris-HCl (pH 8.5) and the box was immediately sealed and stored at roomtemperature. Minor variations in this procedure such as altering bufferconditions are within the scope of the present invention. For example,in a preferred embodiment of the present invention, buffer conditionswere altered to include 150 mM MgCl₂ and 24% PEG 4000.

After 3-5 days, small microcrystals having a size of 0.1×0.1×0.05 mmappeared in each well. These microcrystals were isolated and redissolvedin 25 microliter of 50 mM Tris-HCl and allowed to stand at roomtemperature. The purified M-CSF crystallized from solution into largehexagonal prism shaped crystals ranging in size from 0.3×0.3×0.3 mm to 1mm×2 mm×0.5 mm. These crystals were stable at room temperature for atleast three months. In some instances, an artificial mother liquor wasprepared using 23% PEG 4000 and 150 mM MgCl₂ crystals were then added tothis mother liquor. In these cases, the crystals were removed from themother liquor immediately prior to analysis.

Using reducing and non-reducing SDS-PAGE analysis, the M-CSF in thecrystals was shown to be identical in molecular weight to thebiologically active starting material. Thus, the M-CSF structureobtained from the crystals is likely to be essentially identical to thestructure of biologically active M-CSF.

EXAMPLE 3

Glass microscope slides were prepared as described in Example 2. 7microliters of the same concentrated M-CSFNe protein solution was mixedin each well of the spot plate with 7 microliter of 30% (v/v) PEG 4000,0.2M ammonium acetate, 0.1M acetate buffer (pH 7.5). The spot plate wasthen placed in a clear plastic sandwich box containing 20 ml of 30% PEG4000, 0.2M ammonium acetate, 0.1M acetate buffer (pH 7.5) and the boxwas immediately sealed and stored at room temperature. After 3-5 days,thin, plate-like, fragile crystals having a size of approximately0.3×0.3×0.05 mm appeared.

EXAMPLE 4 Preliminary X-ray Analysis

X-ray crystallographic analysis using precession photographs showed thatthe crystals produced in Example 2 have an orthorhombic crystal latticein the P2₁ 2₁ 2₁ space group with cell dimensions a=33.54, b=65.26,c=159.63, d=90.0, c=90.0 and f=90.0 angstroms and diffract to a nominalresolution of 2.6 angstroms using synchrotron radiation. These dataprovided a unit cell volume of 348084.5 angstroms³, which is consistentwith a dimer in the crystallographic asymmetric unit with 52% of theunit cell volume being occupied by solvent. FIG. 1 is a 12-degreeprecession photograph of the Okl-zone section of the M-CSF crystal. Thephotograph was taken using a precession camera manufactured byEnraf-Nonius Company (Delft, Holland), mounted on a Rigaku RU-200 X-raygenerator operated at 50 kV and 50 mA.

EXAMPLE 5 Testing of M-CSF Receptor Binding Ability Using Soluble M-CSFReceptor

An essential step in the biological function of M-CSF in vivo is thebinding to the M-CSF receptor, also referred to as the c-fims geneproduct. Recombinant human soluble M-CSF receptor (rhsM-CSFR),representing amino acids 20 to 511 (Coussens, L et al., Nature, 320:277(1986)) was used as an in vitro assay reagent to test thereceptor-binding ability of M-CSF proteins. To generate a soluble formof the transmembrane receptor, only the extracellular domain of thehuman M-CSF receptor was expressed in a baculovirus/insect cellrecombinant expression system. In order to purify the soluble receptorwithout adversely effecting tertiary or quaternary structure,non-denaturing chromatographic methods were chosen, as described below.Other choices exist for the purification of the recombinant receptor.Affinity chromatography may be employed when either a suitable antibodyto or ligand for the receptor are available. Alternatively, "tags" maybe added to the C-terminus of the recombinant receptor, i.e., KT3antibody recognition sequence, and purified by an anti-tag antibody,i.e., KT3, column, for use in affinity chromatography. In expressionsystems in which the rhsM-CSFR is glycosylated, lectin chromatographycan be used to enrich for specific glycoproteins.

The rhsM-CSFR can be used to study ligand/receptor interactions as wellas ligand-induced receptor dimerization. The assay used to detectligand/receptor binding employed the use of size exclusion-HPLC,essentially as described in European Patent Application WO92/21029, C.Cunningham, et al., with the following modifications: the column usedwas a Superose 6 (Pharmacia LKB Biotechnology, Inc.) and the mobilephase was PBS at 0.5 ml/min and a M-CSF to rhsM-CSFR ratio of 1:2. Atthis ratio, the M-CSF/rhsM-CSFR complex chromatographed with an apparenthydrodynamic radius of 190,00 molecular weight, the molecular weightexpected for a M-CSF(rhsM-CSFR)₂ complex. Other assays may be employedto measure ligand/receptor binding or receptor dimerization such aschemical crosslinking and SDS-PAGE or immunoprecipitation and SDS-PAGE.Molecules that inhibit receptor dimerization but not ligand bindingprovide another method to antagonize M-CSF actions.

The DNA encoding rhsM-CSFR was cloned for expression in insect cellsusing the following general strategy. The portion of the c-fms genecorresponding to amino acids one to 511 was amplified from humanmacrophage cDNA by polymerase chain reaction (PCR) using an upstreamprimer of: 5'-GCGTACCATGGGCCCAGGAGTTCTGC-3' (SEQ ID NO. 9) and adownstream primer of: 5'-AGTCGAGGATCCTCAATCCGGGGGATGCGTGTG-3' (SEQ IDNO. 10). The underlined sequences are the NcoI and BamHI restrictionsites used to subclone the PCR product into the pAcC5 vector (Luckov etal., Bio/Technology 6:47-55). The pAcC5:hsM-CSFR vector was expressed inSF9 insect cells using a baculovirus helper vector as previouslydescribed (Summers, et al., A Manual of Methods for Baculovirus Vectorsand Insect Cell Culture Procedures (1987)).

Approximately two liters of serum-free 72-hour conditioned medium wascollected by centrifugation and filtration from SF9 cells infected withpAcC5:hsM-CSFR construct described above. The material was diafilteredwith DEAE buffer A 10 mM Tris, pH 8.8, containing the following proteaseinhibitors (which were added all buffers throughout the purification): 1mM EDTA, 2 μg/ml leupeptin and 100 μM PMSF! and concentrated 20-foldwith a 20,000 molecular weight cutt-off Pyrostat UltrafiltrationMembrane (Sartorius). The retentate was loaded onto a DEAE Sepharosecolumn (Pharmacia LKB Biotechnology, Inc., Piscataway, N.J.) having abed volume of 100 ml that had been preequilibrated with DEAE Buffer A.Elution was at 5 ml/min with a 0-0.8M NaCl gradient in 500 ml of DEAEBuffer A. Fractions enriched in rhsM-CSFR were detected Western AnalysisBurnett, R., Anal. Biochem., 112:195 (1981)! and dot blot analysis ofserially diluted fractions, using anti-c-fms monoclonal antibodies(Oncogene Sciences, Inc.). The dot blot assay was used throughout thepurification to identify fractions containing rhsM-CSF. Enrichedfractions were pooled, made 0.8M in ammonium sulfate, adjusted to pH 7.0and loaded onto a Phenyl TSK-5-PW HPLC column (7.5×75 mm) (BioRad). Thecolumn was eluted at 1 ml/min with a decreasing ammonium sulfategradient over 45 minutes, peak fractions were pooled and concentrated10-fold with a stir cell concentrator using a YM30 membrane (Amicon).The retentate was chromatographed with FG3000XL size exclusion column(DU PONT, Wilmington, Del.) using a mobile phase a phosphate-bufferedsaline (PBS) at 3 ml/min. The purified receptor was pooled, concentratedto 1 mg/ml as above and stored at 4° C. This process recovered 650 μg ofrhsM-CSFR, purified 200-fold. The preparation was about 95% homogeneousas assayed by SDS-PAGE stained with Coomassie Blue.

EXAMPLE 6 Crystallization of M-CSF/Soluble M-CSF Receptor Complex

To crystallize the M-CSF/rhsM-CSFR complexes, glass microscope slidesare prepared as described in Example 2. The M-CSF composition used isincubated with a purified soluble form of the M-CSF receptor, truncatedat a residue before the transmembrane region, to form an M-CSF/ receptorcomplex. In certain cases, the rhsM-CSFR is deglycosylated prior to thesize exclusion step by incubation with N-glycanase (Genzyme, CambridgeMass.) according to the manufacturer's instructions. A small quantity ofM-CSF/receptor solution is mixed in each well of the spot plate with acomparable quantity of a drop solution (such as about 20% (v/v) PEG4000, 0.2M MgCl₂, 0.1M Tris-HCl (pH 8.5)). The spot plate is then placedin a clear plastic sandwich box containing a small amount of precipitantsolution (such as about 23% PEG 4000, 0.2M MgCl₂, 0.1M Tris-HCl (pH8.5)). The box is immediately sealed and stored at room temperature.

After a few days, crystalline M-CSF-receptor complex is isolated andredissolved in a solution containing about 50 mM Tris-HCl and is allowedto stand at room temperature. The purified M-CSF-receptor complexcrystallizes from solution to form crystals for X-ray structuralanalysis. To facilitate solution of the crystal structure of suchcomplexes, truncated, non-glycosylated forms of the rhsM-CSFR CSFR(described above) which retain M-CSF binding ability may be employed togenerate M-CSF-receptor complex crystals.

EXAMPLE 7

The biological activity of the non-glycosylated, truncated sequence usedin Examples 2 and 3 was shown to be equal to that of the mature proteinpurified from human urine (Halenbeck, R., et al., Bio/Technology,7:710-715 1989!). As noted, the resulting crystals had an orthorhombiccrystal lattice in the P2₁ 2₁ 2₁ space group, with cell dimensionsa=33.54, b=65.26, and c=159.63 Å. Intensity data were collected usingimaging plates mounted on a Weissenberg camera modified formacromolecular crystallography at the Photon Factory in Tsukuba, Japan.Native data to a nominal resolution of 2.0 Å, and mercury and platinumderivative data were collected using 1.0 Å radiation. Two crystalsettings were used to collect native data Rmerge (I)=7.0%, using allmeasurements with I>0.0!.

Heavy atom derivatives of M-CSF crystals were prepared by soakingcrystals in heavy atom compounds dissolved in the reservoir solution.Isomorphous and anomalous difference Patterson maps clearly revealed onesite for the mercury and two sites for the platinum derivative.Anomalous and isomorphous phase information as used in initial phaserefinement with the PROTEIN program package. The final figure of meritwas 0.62 (8.0-3.0 Å, 6960 reflections). After solvent flattening, B. C.Wang, Methods Enzymol, 115:90 (1985), two bundles of four alpha helicesrelated by an approximate two-fold axis could be seen in the electrondensity map. Rotation and translation parameters of thisnon-crystallographic axis were refined by a density correlation method,J. M. Cox, J. Mol. Biol. 28:151 (1967). Phases were then iterativelyrefined by molecular averaging and solvent flattening, G. Bricogne, ActaCryst., 32:832 (1976), using an envelope calculated by putting 1 Åspheres around all the atoms in the four helical bundle. Chain tracingand model building were done in the resulting map, (using the programFRODO), T. A. Jones, Methods Enzymol. 115:157 (1985), keeping theoriginal MIR map as a reference.

The starting partial model for refinement contained only a polyalaninebackbone for eight helices making up the two bundles. Positionalrefinement using the program XPLOR, A. T. Brunger, J. Mol. Biol. 203:803(1985), gave an R-factor of 0.49 to 3.0 Å. Phase combination with therefined MIR phases resulted in a map of sufficient quality to allow thetracing of two long loops traversing the four helical bundle and a shortloop connecting two of the helices. Two strong peaks in the density, oneat the top of the first helix, and the second lying directly on themolecular two fold axis, were assigned as disulfide bonded cysteines.The number of residues between these two peaks uniquely identified theposition in the sequence of these cysteines and consequently thesequence of the intervening residues. This initial registration wasconfirmed by the presence of a number of regions of strong densitycorresponding to aromatic side chains in the sequence. Partial modelphase combination using the added loops and those side chains that werevisible allowed the remaining residues to be registered, thusdetermining the overall topology of the molecule. The presence of sevendisulfide bonds in the dimer served as important "tether points" toconfirm the correctness of the tracing.

As shown in FIG. 2, the overall topology of this form of M-CSF is thatof an antiparallel four α-helical bundle, in which the helices runup-up-down-down, unlike the more commonly observed up-down-up-downconnectivity of most four helical bundles. A long crossover connectionlinks helix A to helix B and a similar connection is found betweenhelices C and D.

A striking difference from other cytokines and other four helix bundlestructures is that the truncated M-CSFα forms a disulfide-linked dimer,in which the bundles are linked end-to-end, forming an extremely flat,elongated structure (approximate dimensions 85×35×25 Å) as shown inFIGS. 3 and 4A and 4B. There are three intramolecular disulfide bonds ineach monomer (Cys7-Cys90, Cys48-Cys139, Cys102-Cys146) all of which areat the distal end of the molecule. One interchain disulfide bond(Cys31--Cys31) is located at the dimer interface with thenoncrystallographic two-fold symmetry axis passing through it as shownin FIGS. 3 and 4A and 4B. Mutation experiments indicate that all of thecysteine residues in this form of M-CSF may be necessary for fullbiological activity. The structure described herein suggests that theirrole is primarily structural rather than being related to receptorrecognition.

Appendix 1 provides the three-dimensional structure of the truncatedrecombinant M-CSFα dimer as identified by the alpha-carbon positions ofthe amino acid residues in the sequence. The five carboxy terminal aminoacids of each polypeptide of the dimer were not included. As will berecognized to those of skill in the art, the information in Appendix 1is provided in the format used by the Brookhaven Protein Data Bank.

As shown, the molecule has an unusual topology which identifiesimportant regions of M-CSF with regard to M-CSF receptor binding.Specific residues in helices A, C, and D appear to be involved in thespecificity of the interaction. Altering solvent accessible residues inthese regions by site directed mutagenesis to increase or decreaseside-chain interactions with the receptor may be useful to generateM-CSF agonists or antagonists. For example, changing one or morehistidines to non-hydrogen-donor amino acids of similar size may createan M-CSF with altered receptor binding ability.

EXAMPLE 8 Preparation of M-CSF Heterodimers

Purification of M-CSF Monomers:

E. coli harboring the pL-M-CSF vector for NΔ3CΔ158M-CSFα described inU.S. Pat. No. 4,929,700 or NΔ3CΔ221 M-CSFβ C157S, C159S (Kawasaki, etal., in Colony-Stimulating Factors, Dexter, T., Garland, J., and Testa,N., eds. 1990!) were grown in 1 liter of minimal salts medium containingglucose and the appropriate antibiotic. Expression of M-CSF was inducedby shifting the temperature to 39° C. for 4 hr. following the additionof casamino acids to 2%. The cells were harvested by centrifugation andlysed by sonication in 50 mM Tris (pH 8.5), 10 mM EDTA. The cell debriswas recovered by centrifugation, washed with 30% sucrose containing 10mM EDTA, and a portion of the retractile body paste was solubilized in8M urea under reducing conditions. After incubation at 37° C. for 30min., the solubilized M-CSF was clarified by centrifugation andfiltration, and then loaded onto a Bio-Gel TSK-DEAE-5-PW column (7.5×75mm) (BioRad Laboratories, Richmond, Calif.) equilibrated in 8M urea in10 mM Tris (pH 8.5), 5 mM DTT, 0.5 mM EDTA. The monomeric M-CSF waseluted with a 45-min, 0-0.6M NaCl gradient. The M-CSF peak fractionswere pooled and concentrated to 10 mg/ml with a Centricon 10microconcentrator (Amicon).

Formation and Analvsis of Active M-CSF Heterodimers

The M-CSF homodimers were refolded by diluting to a proteinconcentration of 0.5 mg/ml in precooled 50 mM Tris (pH 8.5), 5 mM EDTA,2 mM reduced glutathione and 1 mM oxidized glutathione, and thenincubating at 4° C. The heterodimer was refolded by diluting 158 and221F monomer pools to 1 mg/ml in the same buffer. To monitor therefolding, size exclusion high pressure liquid chromatography (SE-HPLC)analysis was performed by immediately injecting reaction samples onto aG3000SW Ultropack (Pharmacia LKB Biotechnology, Inc., Piscataway, N.J.)column size-exclusion (7.5×600 mm) equilibrated in PBS (pH 6.8).

Fractionated products were analyzed on reducing SDS-PAGE and stainedwith Coomassie, according to the method of Laemmli, Nature (Canada)227:680-685 (1970). Biological activity was determined using the M-CSFdependent NFS-60 bioassay (See Example 10 below). Antibodyneutralization experiments were carried out by pre-incubatingapproximately 5,000 units of M-CSF dimer with varying dilutions of theneutralizing M-CSF 5H410 Mab (made to refolded E. coli CΔ150 M-CSFαdimer) prior to bioassay. (Halenbeck et al, Bio/Technology 7:710 (1989)

The heterodimeric M-CSF product was designed to consist of one chain ofshort clone (from amino acid 4 to 158) and one chain of long clone (fromamino acid 4 to 221). The long-clone chain (221F) also containedsubstitutions of serine for the two non-essential cysteines (at 157 and159) to minimize the possibility of higher-order oligomer formation.

Solubilized refractile bodies of M-CSF 158 and 221F were separatelychromatographed by DEAE-HPLC in 8M urea. Only one major protein peakeluted in each case, and the peak fractions were pooled, based on ananalysis of purity by non-reducing SDS-PAGE and Coomassie staining (datanot shown). The resulting monomer was over 90% pure in each case. Themonomers were separately concentrated to 10 mg/ml, diluted in refoldingbuffer, and refolded at 4° C.

To compare the rates of dimerization of short- and long-clone M-CSF,20μl of each refolding reaction was injected on a SE-HPLC column at 0,2, 18 and 72 hr. The amount of dimeric M-CSF formed was determined fromthe peak area at the molecular weight expected for dimer. In bothrefolding reactions the M-CSF was mostly equilibrated to monomer at t=0and had become about 40% dimeric by 2 hr and nearly 75% dimeric by 18hr. The similarity of the ratio of dimer to monomer between the refolded158 and 221F strongly suggests that the rate of dimer formation is thesame for long- and short-clone M-CSF. Thus, when equal moles of 158 and221F are present in a refolding reaction, the final relative ratios of158 homodimer to 221F homodimer to 158/221F heterodimer are predicted tobe 1:1:2 (Similar distributions have been observed in vivo for isozymesof lactate dehydrogenase.)

Biological Activity of Refolded Homodimers and Heterodimers

The biological activity of the refolded homodimers and heterodimersdescribed above, was examined using the in vitro M-CSF-dependent NFS-60bioassay (See Example 10 below). FIGS. 5A-5C show the result of thesestudies. These SE-HPLC and biological activity profiles analyzed after72 hr of refolding, show that the heterodimer, FIG. 5C, displaysactivity very similar to that of the two homodimers, (FIGS. 5A and 5B).Given that the separation of the heterodimer from the homodimers wasnearly complete, it can be concluded that the heterodimer is fullybiologically active in vitro.

To verify that the M-CSF protein eluting from these columns at thepredicted heterodimeric position (between the two homodimers) actuallydid consist of equal moles of short- and long-clone monomers, analysisof a preparative purification of the 158/221F heterodimer was carriedout. Phenyl-HPLC was performed as described above and was shown tocompletely resolve the heterodimer from the 158 and 221F homodimers, asseen in FIG. 6A.

Preparative Purification of M-CSF Heterodimers:

The refolded M-CSF was adjusted to pH 7.0 with 1 N HCl, and ammoniumsulfate was added to 1.2M. The protein was loaded onto a Bio-GelTSK-Phenyl-5-PW column (7.5×75 mm) (BioRad, Richmond, Calif.)equilibrated in 1.2M ammonium sulfate, 100 mM phosphate (pH 7.0). TheM-CSF was eluted with a decreasing gradient of ammonium sulfate from 40%to 80% buffer B (10 mM phosphate, pH 7.0) in 24 min.

Reducing and non-reducing SDS-PAGE (FIGS. 6B and C) showed that internalcontrols (the 158 and 221F dimers) were purified to approximately 95%homogeneity by this column, and each consisted of the single expectedmonomeric band. The gel analysis also showed that the heterodimer waspurified to approximately 95% homogeneity and that it consisted ofequivalent amounts of 158 and 221F monomers. Recovery of purified158/221F heterodimer from refractile body paste to final product, wasgreater than 15%.

The bioactivity of the dimeric M-CSF species was determined and, whencompared to the A₂₈₀ profile in FIG. 6A, confirms the finding that theheterodimer is fully active. The specific activity of the 158/221Fheterodimer. calculated using the peak fraction, was 8.0×10⁷ units/mg,compared to 9.0×10⁷ and 6.8×10⁷ units/mg for 158 and 221F homodimers,respectively.

The biological activity of all three dimer species was neutralized tothe same extent in serial dilution neutralization experiments using the5H410 M-CSF Mab in the NFS-60 bioassay. This antibody also neutralizes"naturally refolded" Chinese hamster ovary cell (CHO)-expressed M-CSF ina similar fashion. This result further suggests that the refoldedconformation of the new M-CSF heterodimer is essentially native-like, atleast with regard to the region within the first 150 amino acids that isresponsible for in vitro activity.

EXAMPLE 9 Selection of Amino Acid Substitutions in M-CSF Based onCrystallographic Data

The X-ray crystallographic data described above provided sufficientstructural information regarding M-CSF to be able to identify a limitedsubset of the amino acids in the protein that are likely to be crucialfor M-CSF receptor binding and biological activity and thus whichrepresented likely candidates for mutagenesis with the ultimate goal ofproviding M-CSF muteins having altered biological activity (i.e.,agonists or antagonists). Based on this information, several criteriawere used to generate a list of possible target amino acids forsubstitution.

The first criterion was solvent exposure or solvent accessibility, whichrefers to amino acids residues at the surface of the protein. Residueshaving a solvent accessible surface area of greater than about 0.25 andpreferably greater than about 0.4 are preferred based on normalizationof the surface area of the amino acid accessible when in the trypeptidegly-x-gly (Kabsch, W. et al., Biopolymers 22:2577 (1983)). Residues werechosen which do not interact with other parts of the protein such as thedimer interface in order to maintain the relative orientation ofmonomers and to avoid disturbing the process of protein folding. Stillanother criterion used in certain instances in selecting candidate aminoacid substances is the relationship of the residues to correspondingresidues in mouse M-CSF. Another important selection criterion was thatthe substitutions be non-conservative so as to attempt to disruptpossible hydrogen bonding or hydrophobic interactions with M-CSFreceptor residues.

Table 1 lists exemplary amino acid residues and exemplary substitutions.Using the criteria for selecting candidates for substitutions set forthabove, those of ordinary skill in the art may readily ascertain otherpossible candidates for substitution.

                  TABLE I    ______________________________________    Candidate Substitutions    Wild Type Amino    Acid and Location   Substitutions    ______________________________________    His (H)      15         Ala(A) or Leu(L)    Gln (Q)      17         Ala(A) or Glu(E)    Gln (Q)      79         Ala(A) or Asp(D)    Arg (R)      86         Glu(E) or Asp(D)    Glu (E)     115         Ala(A)    Glu (E)      41         Lys(K) or Arg(R)    Lys (K)      93         Ala(A) or Glu(E)    Asp (D)      99         Lys(K) or Arg(R)    Leu (L)      55         Gln(Q) or Asp(N)    Ser (S)      18         Ala(A) or Lys(K)    Gln (Q)      20         Ala(A) or Asp(D)    Arg (R)      21         Ala(A), Glu(E),                            or Asp(D)    Ile (I)      75         Lys(K) or Glu(E)    Val (V)      78         Lys(K) or Arg(R)    Leu (L)      85         Glu(E) or Asn(N)    Asp (D)      69         Lys(K) or Arg(R)    Asn (N)      70         Ala(A) or Glu(E)    His (H)      9          Ala(A) or Asp(D)    Asn (N)      63         Lys(K) or Arg(R)    Thr (T)      34         Gln(Q) or Lys(K)    ______________________________________

It is not expected that every candidate substitution listed will resultin the production of M-CSF agonists or antagonists (see Example 12below). Rather they represent a non-exclusive list of candidates likelyto result in the production of agonists or antagonists based on theselection criteria set forth above. It should also be noted that even ifa variant does not act as an agonist or antagonist when compared withnative M-CSF, the variant is still useful for conventional uses of theligand (if it retains the same activity as the ligand) or as forexample, a diagnostic reagent.

EXAMPLE 10 Preparation of H9A, H15A M-CSF Muteins

A variety of M-CSF muteins with altered solvent-accessible residues fromregions of the M-CSF mature N terminus and helices A, C, and D wereconstructed using techniques known in the art. For example, twohistidines in the N-terminal/A helix region were changed to alaninethrough site-directed mutagenesis of a truncated form of M-CSFα (encodedby pLCSF158A). Involvement of one of three M-CSF histidine residues inM-CSF receptor interaction was implicated by our observation thatdiethylpyrocarbonate (DEPC) modification of histidines in M-CSF at a1:100 DEPC:histidine ratio (as described in Meth. in Enzymol. 47:431(1977)) significantly reduced bioactivity.

Plasmid DNA pLCSF158A was prepared from the E. coli strain HW22 carryingthe plasmid pLCSF158A (U.S. Pat. No. 4,929,700, Example 6, "E. colistrain HW22 transformed with pJN653 containing the asp₅₉ SCSF/NΔ3CΔ158gene"). The strain was grown in 350 ml R2 media (2× Luria Brothcontaining 1% sodium chloride and no glucose, J. Bact., 74:461 (1957))containing 50 micrograms/ml ampicillin at 30° C. with shaking overnight.Plasmid DNA was prepared from the cells using a Qiagen-tip 100 columnaccording to the manufacturer's directions.

Twenty micrograms of pLCSF158A DNA were digested with 66 units ofHindIII and 66 units of StuI at 37° C. for 3 hr. 20 min. in 200microliters 1× New England Biolabs NEBuffer 2 (New England Biolabs,Beverly, Mass.). The DNA was extracted with phenol and chloroform, thenethanol precipitated. The DNA was treated with one unit of CalfIntestinal Alkaline Phosphatase in 100 microliters of 1×Dephosphorylation Buffer, supplied by Boehringer Mannheim (Indianapolis,Ind.), at 37° C. for 30 min. An additional unit of Calf IntestinalAlkaline Phosphatase was added to the reaction and incubation wascontinued at 50° C. for 1 hr.

The resulting DNA was then run on a 1% FMC Bioproducts (Rockland, Me.)Sea KEM® GTG® agarose gel. The 5.7 kb pLCSF158A fragment was cut fromthe gel and purified on Qiagen (Chatsworth, Calif.) Qiaex beadsaccording to the manufacturer's directions.

Polymerase chain reaction (PCR) was then performed and a PCR product wasproduced that contained a mutagenized M-CSF sequence in which histidines9 and 15 (counting from the mature N-terminus) were altered to alanine(generating an H9A, H15A PCR fragment). The 5' portion of the M-CSF genewas amplified from the plasmid pLCSF158A in a PCR reaction using theprimers LF73 and LF74. Details of PCR are provided by Mullis, K. et al.,U.S. Pat. No. 4,683,202; Ehrlich, H., U.S. Pat. No. 4,582,788; Saiki etal., U.S. Pat. No. 4,683,195; Mullis, K. B., Cold Spring Harbor Symp.Quant. Biol. 51:263-273 (1986); Saiki et al., Bio/Technology3:1008-10012 (1985); and Mullis, K. B. et al., Meth. Enzymol 155:335-350(1987), all of which are incorporated herein by reference. The sequencesof these primers are:

LF73:

5'-AGGTGTCTCATAGAAAGTTCGGACGCAGGCCTTGTCATGCTCTTCATAATCCTTGG-3' (SEQ IDNO. 1)

LF74:

5'-CAGGAGAAAGCTTATGTCTGAATATTGTAGCGCCATGATTGGGAGTGGAGCCCTGCAG-3' (SEQ IDNO. 2)

The expected PCR product was designed to include 337 bp of pLCSF158Asequence, with the HindIII and StuI sites located at each end of theproduct for cloning, and the histidine codons for His 9 and His 15, CAC,mutated to an alanine codons, GCC.

This product was amplified in four separate PCR reactions eachcontaining 100 ng of pLCSF158A DNA, 50 pmoles LF73, 50 pmoles LF74, 37.5μM dNTPS, 5% glycerol, 1× Perkin Elmer Cetus PCR Buffer, and 2.5 unitsof Perkin Elmer Cetus AmpliTaq® DNA Polymerase in a 100 microlitervolume. The amplification was carried out in a Perkin Elmer Cetus DNAthermocycler. Before adding the AmpliTaq®, the reactions were brought to95° C. The amplification was carried out for 25 cycles ramping to adenaturation temperature of 95° C. in 1 sec., denaturing at 95° C. for 1min.; ramping to an annealing temperature of 68° C. in 1 sec., annealingat 68° C. 1 min.; ramping to an extension temperature of 72° C. in 30sec., extending at 72° C. 1 min., 30 sec. Final extension was carriedout at 72° C. for 10 min.

Five microliters of each reaction were run on a 3% agarose gel (1.5% FMCBioproducts SeaKem® GTG® agarose, 1.5% FMC Bioproducts NuSeive® GTG®agarose in Tris-Borate buffer) (FMC Bioproducts, Rockland, Me.). Gelswere then stained with ethidium bromide. For each reaction, a major bandwas visible at approximately 337 bp.

The four reactions were pooled, extracted with phenol and chloroform,precipitated with ethanol, resuspended and digested with 250 units ofStuI in a final volume of 500 microliters 1× NEBuffer 2 at 37° C. for 2hr., 500 units of HindIII were added to the reaction, the volumeincreased to 1 ml in 1× NEBuffer 2 and digestion was continued at 37° C.for an additional 2.5 hr. The DNA was electrophoresed on a 3% agarosegel. The 300 bp digested product was cut from the gel and purified onQiagen Qiaex beads according to the manufacturer's directions.

Approximately 68 ng of the HindIII/StuI digested PCR product was thenligated to approximately 28 ng of the 5.7 kb HindIII/StuI digestedpLCSF158A vector DNA at an insert-to-vector ratio of approximately 5:1.Ligation was carried out with 1 unit of Boehringer Mannheim T4 DNAligase in 1× ligation buffer, supplied by the manufacturer, in a20-microliter volume at 16° C. overnight. As a control 28 ng of the 5.7kb HindIII/StuI digested pLCSF158A vector DNA was ligated to itselfunder the same conditions with no insert present.

Half of each ligation mixture was used to transform competent E. coliDG116 (ATCC# 53606) cells using a protocol similar to the calciumchloride procedure described in Molecular Cloning a Laboratory ManualManiatis et al., Cold Spring Harbor Laboratory (1982). Transformed cellswere allowed to express at 30° C. with no selection for 90 min., platedon R2-4 (10 g tryptone, 5 g yeast extract, 5 g NaCl, 2 drops antifoam A,4 ml 50% glucose and 15 g agar in 1 liter) plates containing 50micrograms/ml ampicillin. The plates were incubated at room temperature72 hr. One fourth of each transformation was plated. For the ligationcontaining the insert, 66 ampicillin resistant colonies appeared on theplates. For the ligation with no insert, no colonies appeared.

One of these colonies, designated strain TAF172-2, was picked andcultured in 350 ml R2 broth with 50 micrograms/ml ampicillin at 30° C.with shaking overnight. A frozen stock in 40% glycerol was made fromthis culture and stored at -70° C. DNA was isolated from the cultureusing Qiagen-tip 100 columns as described above.

The purified DNA, pTAF172-2, was sequenced using the di-deoxy method andshown to contain the sequence of pLCSF158A coding for M-CSF NΔ3CΔ158with His 9 and His 15 mutated to encode Ala.

The M-CSF mutein NΔ3CΔ158 H9A, H15A encoded by pTAF172-2 was expressed,purified, refolded to form dimeric protein and assayed essentially asdescribed in U.S. Pat. No. 4,929,700 Example 5, using 8M urea as adenaturant and in the DEAE purification step.

The N-terminal sequence of the purified mutein was determined through 20cycles, using a standard automated Edman degradation method, and shownto be identical to that of the parental NΔ3CΔ158 M-CSFα referenceprotein except that His 9 and His 15 had been altered to Ala. Proteinconcentration was determined using A280 and an extinction co-efficientof 0.68.

The purified mutein dimers were subjected to bioassay using NFS-60 cellswhich is an M-CSF dependent cell line which forms colonies in thepresence of active M-CSF. Standards and purified mutein samples wereserially diluted 1:2 in RPMI media (10% fetal bovine serum) in a 50microliter volume in 96-well microtiter plates. 50 microliters NFS-60cells (ATCC NO. CRL 1838), maintained in 4000U/ml M-CSF, washed 2×, anddiluted to a concentration of 1×105 cell/ml, were added to each samplewell. Plates were incubated at 37° C., 5% CO₂ for 44 h. 5 mg/ml MTT dyewas added to each sample and incubation continued for 4 h. 20% SDS wasadded to each sample. The plates were wrapped in foil and leftovernight. The absorbance of each sample was read at 570 nm and comparedto a standard of known M-CSF concentration. The H9A, H15A mutein showeda specific activity of 7.6×10³ U/mg compared to 6.9×10⁷ U/mg for theparental M-CSF NΔ3CΔ158 reference in the same assay. This represents anearly 10,000 fold reduction in biological activity for the mutein. Thesame M-CSF mutein preparation was shown to have greatly decreased M-CSFreceptor-binding ability using the NFS-60 receptor competition assaydescribed in this example. Because the H9A, H15A M-CSF mutein wasotherwise not significantly different from the parental M-CSFα,including crystallizability and space group (see Example 12) we believethe decrease in biological activity is not due to gross deformation ofstructure but reflects an alteration in important M-CSF receptorcontacts.

Using essentially the same methodology, two M-CSF muteins contactingsingly substituted histidines at residues 9 and 15 were generated (e.g.H9A and H15A). The H9A construct utilized LF80:

5'-CAGGAGAAAGCTTATGTCGAATATTGTAGCGCCATGATTGGGAGTGGACACCTGCAG-3' (SEQ IDNO 3); and LF73 (described in this example) as the PCR primers. The H15Aconstruct utilized LF81:

5'-CAGGAGAAAGCTTATGTCTGAATATGTAGCCACATGATTGGGAGTGGAGCCCGCAG-3' (SEQ IDNO. 4); and LF 73 (described in this example) as PCR primers. Biologicalassay of the purified muteins immediately following the refolding stepdescribed above showed approximate biological specific activities asfollows: 4×10⁶ U/mg for H9A and less than 3×10³ U/mg for H15A, in anassay which the parental M-CSF construct displayed 8×10⁷ U/mg. Thisinformation, combined with that described above, suggests that H15A aswell as possible H9A represents contacts that are important for M-CSFreceptor binding. Nearby solvent accessible residues such as Y6 and S13(see also Table 1) may also represent M-CSF receptor contact residues.Non-proteinaceous mimics of the side chains of H9, H15, and nearbysolvent accessible side chains may represent M-CSF agonists orantagonists. Such residues should be left unchanged in M-CSF muteinconstructs designed to retain full M-CSF receptor binding but to haveM-CSF antagonist properties because they lack significant M-CSFbioactivity. Homodimers of muteins that retain full receptor-bindingability and display significantly reduced bioactivity should representM-CSF antagonists. M-CSF muteins that are greatly decreased in bothM-CSF bioactivity and receptor binding ability (such as H15A) maygenerally be useful in M-CSF immunoassay applications and mightrepresent useful therapeutic agents for patients having auto-antibodiesto M-CSF.

EXAMPLE 11 Preparation of Q20A, V78K M-CSF Muteins

Using essentially the same methodology described in Example 10, a doublemutant of M-CSF (Q20A, V78K) was constructed to test the importance ofsolvent accessible residues in the central portion of helices A and C.The following PCR primers were used.

LF63:

5'-AGGAGAAAGCTATGTCTGAATATGTAGCCACATGATGGGAGTGGACACCTGCAGTCCTGGCTCGGCTG-3' (SEQ ID NO. 5)

LF64:

5'-GGACGCAGGCCTTGTCATGCTCTTCATAATCCTGGTGAAGCAGCTCTTCAGCCTCAAAGAGAGTCCTGCAGCTGTAATGGC-3' (SEQ ID NO. 6)

The resulting mutein was expressed, refolded, purified, and assayed asdescribed in Example 8. The specific biological activity was 1.4×10⁷U/mg, approximately 8-10 fold lower than that of the parental M-CSFαreference standard. The receptor binding activity of this mutein wasalso decreased.

This result again supports the prediction of over crystallographic studyof truncated M-CSFα which concluded that important M-CSF receptorcontact residues exist among the solvent accessible residues in helicesA and/or C and/or D. Certain of these mutations will, as we have shown,have lower biological activity and lower M-CSF receptor-binding ability.Some may have lower biological activity without a decrease inreceptor-binding ability. Some may have increased biological activityand receptor binding ability, and some may have no affect on either.

Two examples of the latter are Q17A, R21A (produced using PCR primersLF72:

5'-TTGTAGCCACATGATTGGGAGTGGACACCGGCGTCCTGCAGGCGCGATTGAC-3' and LF73(described in Example 9) and E115A, N119A (produced using LF75:

5'-CATGACAAGGCCTGCGTCCGAACCTATGAGACACCTCTCCAGTTGCGGCGAAGGTCAAGGCTGTTTAATG-3' (SEQ ID NO. 7); and

LF79: 5'-GGATCAGGATCCCTCGGACTGCCTC-3' (SEQ ID NO. 8)).

Both of these constructs changed side chain properties ofsolvent-accessible amino acids in the areas of interest but did notaffect biological specific activity, compared to the parental referencemolecule. These results indicate that residues Q17, R21, E115, and N119do not need to be altered in muteins designed to have M-CSF agonist orantagonist activity. In fact, to minimize the likelihood of antibodyformation to potentially administered M-CSF-based proteinaceous drugs,it is desirable to retain the solvent-accessible parental M-CSF residues(to resemble the native molecule) whenever possible.

The retained activity of the muteins including changes at Q17, R21,E115, and N119 does not rule out large affects on activity contributedby nearby residues (such as H15). In fact, the regions we have alteredare predicted by the crystal structure to be important for receptorbinding and/or signaling. Antagonistic M-CSF muteins may require use ofmultiple residue changes per mutein or use of heterodimeric moleculescontaining one or more mutations in each polypeptide chain, since M-CSFresidues important in receptor signaling are believed to be composed ofdiscontinuous regions of M-CSF.

EXAMPLE 12 Formation of M-CSF Heterodimers Having DecreasedReceptor-Binding Ability and/or Decreased Biological Specific Activity

M-CSF can be folded in vitro to generate fully active heterodimers, asshown in Example 8. By making heterodimers of M-CSF which incorporateM-CSF muteins with altered M-CSF signaling ability, it should bepossible to generate antagonists of M-CSF useful for treatment ofpatients with M-CSF mediated diseases. To generate a heterodimercontaining one subunit of M-CSFα NΔ3CΔ158 H9A, H15A and one subunit ofM-CSFβ NΔ3CΔ221 C157S, C159S, each mutein was expressed in E. coli andpurified separately by DEAE Sepharose under denaturing and reducingconditions as described in Example 8. The two muteins subunits weremixed together prior to refolding to generate a solution containing afinal mutein molar ratio of 1:1, then this solution was diluted to 0.2mg/ml with refolding buffer as described in Example 8. Followingrefolding, the heterodimeric molecule was separated from the homodimersby two consecutive passes over a Phenyl TSK-5-PW HPLC column asdescribed in Example 8. No contaminants were detected when the purifiedheterodimer preparation was examined by non-reduced SDS-PAGE or sizeexclusion HPLC using a BioSil SEC250 column (BioRad).

The purified heterodimer was submitted to the NFS60 cell based bioassaydescribed in Example 8. The calculated specific activity was 2.9×10⁶U/mg which correlated to a 35-fold reduction as compared to the activityof the parental M-CSF heterodimer described in Example 10. The relativebinding affinity to cell surface M-CSF receptor was measured byradioligand displacement in which the displacement of ¹²⁵ I-M-CSF froman M-CSF receptor by an M-CSF mutein was measured using methods wellknown in the art. In brief, the following were added in a final volumeof 100 μl in each well of a 96-well cell culture plate: approximately80,000 cpm of purified recombinant human M-CSF labeled with ¹²⁵ I (usingIodobeads as described by the manufacturer, Pierce, Rockford, Ill.),300,000 NFS-60 cells that had been washed and then grown for 18 hours ingrowth medium minus the normal maintenance level of M-CSF, plusunlabeled M-CSF that had been serially diluted in the same medium. Theplates were incubated at 4° C. for 20 hours and the cells were collectedon glass-fiber filters. Maximum binding was measured in the absence ofunlabeled M-CSF and non-specific binding was measured in the presence of1000-fold greater concentration of unlabeled M-CSF (compared to labeledM-CSF). The concentration of M-CSF required to inhibit 50% of thelabeled M-CSF binding to the cells (IC₅₀) was used to determinedifferences in affinity. Results are expressed as percent displacementof radioactive M-CSF versus mutein concentration (FIG. 7). The IC₅₀ ofthe heterodimer (FIG. 7 closed squares) was increased 30-fold to about500 pM as compared to an IC₅₀ of about 17 pM for M-CSFα NΔ3CΔ158 (158)(FIG. 7 closed circles). The similarity between the reduction inspecific activity and receptor affinity of the heterodimer indicatesthat the reduction in bioactivity was due to decreased receptor-bindingability. Similarly, the binding affinities of the Q20A,V78KF (FIG. 7open circles) and H9A,H15A (open squares) muteins were also measured inthis radioligand displacement assay. The Q20A,V78K mutein had an IC₅₀ ofabout 100 pM and the H9A,H15A mutein has an IC₅₀ of about 1 μM,correlating to decreased binding affinities of 5-fold and 50,000-fold,respectively. For each mutein, the reduction in receptor affinity wassimilar to the reduction in specific activity, again indicating that thereduction in bioactivity was due to reduced receptor-binding ability.

EXAMPLE 13 Crystallization and Characterization Of M-CSF H9A, H15AMuteins

The H9A, H15A mutein described in Example 10 was crystallized using thehanging drop method described in Examples 1 and 2 using the followingbuffer conditions: 30% polyethylene glycol 4000; 100 mM Li₂ SO₄ ; and100 mM Tris pH 8.5. The crystals produced under these conditions wererhombohedral prisms having dimensions of 0.7 mm×0.2 mm×0.2 mm. X-raycrystallographic analysis using precession photographs showed crystalsin the P2₁ 2₁ 2₁ space group with cell dimensions of a=33.99, b=65.37,c=159.90, d=90, e=90, and f=90 angstroms and diffract to a nominalresolution of 3 angstroms. These physical properties are essentially thesame as those observed for the parental NΔ3CΔ158 M-CSFα molecule andsuggests that the biological effects of the H9A, H15A alterations arenot the consequence of gross global alterations in M-CSF structure, butrather are the result of altered side chains that are important ininteracting with the M-CSF receptor. Alteration of those histidine sidechains may have affected receptor binding by changing atoms thatinteract with, stabilize or facilitate receptor binding or changes inreceptor conformation. Changes such as H15A may also have affected thesefunctions by altering the position of the nearby side chain in M-CSF,most likely in the A and/or C helix regions.

The foregoing examples are presented by way of example and are notintended to limit the scope of the invention as set forth in theappended claims.

                                      APPENDIX 1    __________________________________________________________________________    ATOM         10 CA SER  4  63.753                            80.590                               222.385                                   1.00                                       58.89    ATOM         13 CA GLU  5  64.883                            77.664                               219.972                                   1.00                                       59.56    ATOM         23 CA TYR  6  62.285                            76.840                               217.324                                   1.00                                       54.54    ATOM         37 CA CYS  7  63.509                            80.109                               215.834                                   1.00                                       54.96    ATOM         44 CA SER  8  66.853                            79.529                               214.160                                   1.00                                       54.41    ATOM         52 CA HIS  9  65.466                            77.798                               211.053                                   1.00                                       55.11    ATOM         65 CA MET  10 61.857                            78.767                               211.073                                   1.00                                       50.66    ATOM         74 CA ILE  11 62.173                            80.905                               207.970                                   1.00                                       48.31    ATOM         83 CA GLY  12 63.487                            78.618                               205.354                                   1.00                                       52.83    ATOM         88 CA SER  13 64.609                            79.967                               201.952                                   1.00                                       53.13    ATOM         96 CA GLY  14 61.665                            78.009                               200.514                                   1.00                                       46.05    ATOM         101            CA HIS  15 59.455                            80.924                               201.933                                   1.00                                       40.25    ATOM         114            CA LEU  16 62.210                            83.200                               200.992                                   1.00                                       41.58    ATOM         123            CA GLN  17 62.153                            82.266                               197.328                                   1.00                                       46.17    ATOM         135            CA SER  18 58.378                            81.868                               197.227                                   1.00                                       46.55    ATOM         143            CA LEU  19 58.280                            85.544                               198.199                                   1.00                                       44.75    ATOM         152            CA GLN  20 60.814                            86.044                               195.458                                   1.00                                       39.32    ATOM         164            CA ARG  21 58.457                            84.718                               192.736                                   1.00                                       36.91    ATOM         181            CA LEU  22 55.610                            86.694                               194.252                                   1.00                                       38.27    ATOM         190            CA ILE  23 57.676                            89.822                               193.465                                   1.00                                       34.84    ATOM         199            CA ASP  24 58.677                            88.274                               190.086                                   1.00                                       31.50    ATOM         208            CA SER  25 55.086                            87.802                               188.978                                   1.00                                       31.73    ATOM         216            CA GLN  26 54.284                            91.540                               189.154                                   1.00                                       33.24    ATOM         228            CA MET  27 53.961                            93.634                               186.082                                   1.00                                       34.82    ATOM         237            CA GLU  28 56.227                            96.566                               186.089                                   1.00                                       36.54    ATOM         247            CA THR  29 53.758                            99.303                               186.920                                   1.00                                       44.80    ATOM         256            CA SER  30 54.590                           102.747                               188.323                                   1.00                                       49.40    ATOM         264            CA CYS  31 51.427                           103.423                               190.046                                   1.00                                       43.03    ATOM         271            CA GLN  32 52.166                           104.336                               193.613                                   1.00                                       33.74    ATOM         283            CA ILE  33 50.430                           102.961                               196.634                                   1.00                                       32.48    ATOM         292            CA THR  34 50.240                           104.213                               200.133                                   1.00                                       38.94    ATOM         301            CA PHE  35 51.291                           102.173                               203.199                                   1.00                                       39.16    ATOM         313            CA GLU  36 52.707                           102.849                               206.761                                   1.00                                       32.09    ATOM         323            CA PHE  37 56.073                           101.358                               207.299                                   1.00                                       25.78    ATOM         335            CA VAL  38 59.082                           101.751                               209.490                                   1.00                                       34.46    ATOM         343            CA ASP  39 61.044                           104.798                               208.714                                   1.00                                       44.03    ATOM         352            CA GLN  40 64.648                           103.690                               208.314                                   1.00                                       54.20    ATOM         364            CA GLU  41 65.924                           107.142                               209.332                                   1.00                                       52.01    ATOM         374            CA GLN  42 63.934                           107.629                               212.631                                   1.00                                       44.26    ATOM         386            CA LEU  43 64.770                           104.161                               213.955                                   1.00                                       45.54    ATOM         395            CA ALA  44 68.126                           102.952                               212.789                                   1.00                                       51.53    ATOM         401            CA ASP  45 69.175                           100.197                               215.232                                   1.00                                       47.92    ATOM         410            CA PRO  46 68.861                            97.054                               213.098                                   1.00                                       45.27    ATOM         417            CA VAL  47 67.352                            94.587                               215.613                                   1.00                                       39.92    ATOM         425            CA CYS  48 64.862                            97.220                               216.692                                   1.00                                       36.44    ATOM         432            CA TYR  49 64.089                            98.158                               213.105                                   1.00                                       37.39    ATOM         446            CA LEU  50 63.436                            94.474                               212.189                                   1.00                                       36.15    ATOM         455            CA LYS  51 61.603                            94.029                               215.472                                   1.00                                       38.66    ATOM         468            CA LYS  52 59.322                            96.862                               214.367                                   1.00                                       41.72    ATOM         481            CA ALA  53 59.229                            96.177                               210.534                                   1.00                                       35.81    ATOM         487            CA PHE  54 58.096                            92.701                               211.432                                   1.00                                       40.82    ATOM         499            CA LEU  55 55.236                            93.733                               213.614                                   1.00                                       42.59    ATOM         508            CA LEU  56 54.205                            95.869                               210.697                                   1.00                                       44.24    ATOM         517            CA VAL  57 54.596                            93.257                               207.992                                   1.00                                       35.10    ATOM         525            CA GLN  58 51.646                            91.431                               209.628                                   1.00                                       39.91    ATOM         537            CA ASP  59 49.277                            94.254                               208.663                                   1.00                                       39.60    ATOM         546            CA ILE  60 50.755                            94.729                               205.185                                   1.00                                       37.05    ATOM         555            CA MET  61 50.303                            90.932                               204.676                                   1.00                                       33.86    ATOM         564            CA GLU  62 46.766                            90.931                               206.028                                   1.00                                       48.10    ATOM         574            CA ASP  63 45.602                            94.198                               204.286                                   1.00                                       49.51    ATOM         583            CA THR  64 47.659                            95.244                               201.187                                   1.00                                       42.65    ATOM         592            CA MET  65 49.106                            92.155                               199.603                                   1.00                                       37.90    ATOM         601            CA ARG  66 45.646                            91.249                               198.376                                   1.00                                       39.50    ATOM         618            CA PHE  67 45.516                            88.245                               195.824                                   1.00                                       35.13    ATOM         630            CA ARG  68 42.655                            86.013                               194.326                                   1.00                                       44.68    ATOM         647            CA ASP  69 41.727                            83.454                               196.865                                   1.00                                       47.42    ATOM         656            CA ASN  70 43.117                            80.057                               195.984                                   1.00                                       44.47    ATOM         667            CA THR  71 45.873                            81.088                               193.518                                   1.00                                       34.80    ATOM        676 CA PRO  72 49.607                            80.251                               194.114                                   1.00                                       29.78    ATOM        683 CA ASN  73 50.307                            83.745                               195.418                                   1.00                                       28.47    ATOM        694 CA ALA  74 47.469                            83.922                               197.953                                   1.00                                       23.24    ATOM        700 CA ILE  75 48.690                            80.431                               199.144                                   1.00                                       22.87    ATOM        709 CA ALA  76 52.212                            81.892                               199.669                                   1.00                                       29.47    ATOM        715 CA ILE  77 50.816                            84.796                               201.668                                   1.00                                       37.21    ATOM        724 CA VAL  78 48.902                            82.158                               203.857                                   1.00                                       33.69    ATOM        732 CA GLN  79 52.207                            80.382                               204.466                                   1.00                                       33.36    ATOM        744 CA LEU  80 54.040                            83.518                               205.449                                   1.00                                       32.55    ATOM        753 CA GLN  81 51.152                            84.517                               207.743                                   1.00                                       35.07    ATOM        765 CA GLU  82 51.541                            81.115                               209.443                                   1.00                                       37.26    ATOM        775 CA LEU  83 55.367                            81.348                               209.619                                   1.00                                       35.10    ATOM        784 CA SER  84 54.960                            84.840                               211.066                                   1.00                                       39.64    ATOM        792 CA LEU  85 52.608                            83.541                               213.704                                   1.00                                       42.55    ATOM        801 CA ARG  86 54.984                            80.903                               215.121                                   1.00                                       42.96    ATOM        818 CA LEU  87 57.916                            83.317                               214.676                                   1.00                                       45.11    ATOM        827 CA LYS  88 56.504                            85.876                               217.138                                   1.00                                       50.71    ATOM        840 CA SER  89 57.621                            83.610                               219.825                                   1.00                                       54.58    ATOM        848 CA CYS  90 61.211                            84.524                               219.014                                   1.00                                       46.79    ATOM        855 CA PHE  91 60.235                            88.070                               219.743                                   1.00                                       46.37    ATOM        867 CA THR  92 59.619                            89.812                               223.026                                   1.00                                       52.01    ATOM        876 CA ALA  93 56.916                            92.445                               223.523                                   1.00                                       56.09    ATOM        882 CA ASP  94 57.914                            96.123                               223.998                                   1.00                                       59.35    ATOM        891 CA TYR  95 55.685                            99.100                               225.194                                   1.00                                       66.22    ATOM        905 CA GLU  96 52.401                           100.066                               223.612                                   1.00                                       65.51    ATOM        915 CA GLU  97 53.343                           103.890                               223.182                                   1.00                                       62.60    ATOM        925 CA HIS  98 56.046                           102.712                               220.836                                   1.00                                       58.47    ATOM        938 CA ASP  99 53.422                           100.733                               218.887                                   1.00                                       59.97    ATOM        947 CA LYS 100 52.950                           103.470                               216.162                                   1.00                                       57.00    ATOM        960 CA ALA 101 56.259                           105.270                               216.975                                   1.00                                       48.82    ATOM        966 CA CYS 102 58.861                           105.450                               214.154                                   1.00                                       42.41    ATOM        973 CA VAL 103 56.175                           104.773                               211.621                                   1.00                                       33.87    ATOM        981 CA ARG 104 55.993                           106.851                               208.456                                   1.00                                       42.76    ATOM        998 CA THR 105 53.577                           106.853                               205.501                                   1.00                                       38.00    ATOM        1007            CA PHE 106 54.931                           105.944                               202.136                                   1.00                                       33.48    ATOM        1019            CA TYR 107 53.853                           106.546                               198.455                                   1.00                                       34.28    ATOM        1033            CA GLU 108 55.765                           103.906                               196.543                                   1.00                                       33.88    ATOM        1043            CA THR 109 55.266                           101.309                               193.854                                   1.00                                       36.94    ATOM        1052            CA PRO 110 53.745                            97.857                               194.657                                   1.00                                       29.84    ATOM        1059            CA LEU 111 57.132                            96.374                               193.761                                   1.00                                       35.66    ATOM        1068            CA GLN 112 58.991                            98.756                               196.160                                   1.00                                       33.48    ATOM        1080            CA LEU 113 56.612                            97.691                               198.912                                   1.00                                       24.26    ATOM        1089            CA LEU 114 57.253                            93.932                               198.091                                   1.00                                       30.75    ATOM        1098            CA GLU 115 61.063                            94.385                               197.984                                   1.00                                       39.68    ATOM        1108            CA LYS 116 60.805                            95.824                               201.526                                   1.00                                       39.24    ATOM        1121            CA VAL 117 58.669                            92.840                               202.743                                   1.00                                       32.46    ATOM        1129            CA LYS 118 61.312                            90.620                               201.111                                   1.00                                       37.34    ATOM        1142            CA ASN 119 64.231                            92.316                               202.959                                   1.00                                       39.46    ATOM        1153            CA VAL 120 62.470                            92.033                               206.370                                   1.00                                       32.07    ATOM        1161            CA PHE 121 61.935                            88.268                               206.034                                   1.00                                       26.35    ATOM        1173            CA ASN 122 65.404                            88.049                               204.416                                   1.00                                       36.04    ATOM        1184            CA GLU 123 67.372                            89.816                               207.138                                   1.00                                       40.82    ATOM        1194            CA THR 124 65.331                            88.435                               210.122                                   1.00                                       41.72    ATOM        1203            CA LYS 125 66.456                            85.000                               208.648                                   1.00                                       43.07    ATOM        1216            CA ASN 126 70.010                            86.339                               208.179                                   1.00                                       48.89    ATOM        1227            CA LEU 127 70.000                            87.300                               211.903                                   1.00                                       47.97    ATOM        1236            CA LEU 128 68.315                            84.194                               213.435                                   1.00                                       47.21    ATOM        1245            CA ASP 129 71.086                            82.134                               211.864                                   1.00                                       50.11    ATOM        1254            CA LYS 130 73.448                            84.146                               214.117                                   1.00                                       52.04    ATOM        1267            CA ASP 131 71.442                            84.303                               217.45i                                   1.00                                       49.58    ATOM        1276            CA TRP 132 67.946                            82.685                               218.322                                   1.00                                       53.33    ATOM        1292            CA ASN 133 67.568                            85.178                               221.086                                   1.00                                       59.61    ATOM        1303            CA ILE 134 68.572                            88.447                               219.305                                   1.00                                       49.82    ATOM        1312            CA PHE 135 64.945                            89.516                               219.045                                   1.00                                       54.55    ATOM        1324            CA SER 136 64.745                            89.689                               222.796                                   1.00                                       62.26    ATOM        1332            CA LYS 137 66.563                            93.119                               222.569                                   1.00                                       54.38    ATOM        1345            CA ASN 138 64.785                            95.734                               224.504                                   1.00                                       58.71    ATOM        1356            CA CYS 139 63.902                            98.337                               222.050                                   1.00                                       51.21    ATOM        1363            CA ASN 140 61.704                           100.578                               224.157                                   1.00                                       50.25    ATOM        1374            CA ASN 141 64.219                           103.338                               224.061                                   1.00                                       58.30    ATOM        1385            CA SER 142 65.158                           102.992                               220.352                                   1.00                                       52.18    ATOM        1393            CA PHE 143 61.498                           103.434                               219.645                                   1.00                                       45.87    ATOM        1405            CA ALA 144 61.829                           106.488                               221.831                                   1.00                                       47.49    ATOM        1411            CA GLU 145 64.873                           108.022                               220.050                                   1.00                                       49.94    ATOM        1421            CA CYS 146 62.489                           108.299                               217.017                                   1.00                                       53.65    ATOM        1428            CA SER 147 60.005                           111.197                               217.009                                   1.00                                       57.45    ATOM        1436            CA SER 148 57.163                           110.805                               214.395                                   1.00                                       59.53    ATOM        1444            CA ALA 149 54.284                           108.658                               214.945                                   1.00                                       59.20    ATOM        1450            CA GLY 150 51.655                           108.783                               212.248                                   1.00                                       61.95    ATOM        1455            CA HIS 151 48.764                           106.945                               210.680                                   1.00                                       69.20    ATOM        1468            CA GLU 152 46.415                           107.377                               207.803                                   1.00                                       76.61    ATOM        1478            CA ALA 153 45.813                           106.188                               204.264                                   1.00                                       78.85    ATOM        1492            CA SER 404 43.875                            81.916                               155.536                                   1.00                                       38.68    ATOM        1495            CA GLU 405 41.939                            78.947                               156.798                                   1.00                                       43.57    ATOM        1505            CA TYR 406 44.549                            78.204                               159.422                                   1.00                                       41.97    ATOM        1519            CA CYS 407 43.251                            81.196                               161.434                                   1.00                                       36.99    ATOM        1526            CA SER 408 41.011                            78.917                               163.442                                   1.00                                       34.02    ATOM        1534            CA HIS 409 43.988                            77.122                               165.021                                   1.00                                       40.02    ATOM        1547            CA MET 410 46.155                            79.994                               165.895                                   1.00                                       36.48    ATOM        1556            CA ILE 411 44.849                            80.843                               169.353                                   1.00                                       37.88    ATOM        1565            CA GLY 412 44.508                            77.692                               171.141                                   1.00                                       39.09    ATOM        1570            CA SER 413 43.275                            77.480                               174.756                                   1.00                                       45.21    ATOM        1578            CA GLY 414 46.813                            77.142                               176.120                                   1.00                                       41.00    ATOM        1583            CA HIS 415 47.238                            80.801                               175.208                                   1.00                                       38.01    ATOM        1596            CA LEU 416 44.181                            81.554                               177.458                                   1.00                                       39.12    ATOM        1605            CA GLN 417 45.501                            79.575                               180.501                                   1.00                                       39.79    ATOM        1617            CA SER 418 48.547                            81.846                               180.317                                   1.00                                       36.95    ATOM        1625            CA LEU 419 46.482                            85.106                               180.212                                   1.00                                       33.42    ATOM        1634            CA GLN 420 44.564                            83.708                               183.162                                   1.00                                       38.37    ATOM        1646            CA ARG 421 47.839                            82.875                               185.041                                   1.00                                       41.95    ATOM        1663            CA LEU 422 49.151                            86.514                               184.405                                   1.00                                       33.04    ATOM        1672            CA ILE 423 45.854                            87.864                               185.738                                   1.00                                       25.14    ATOM        1681            CA ASP 424 45.824                            85.549                               188.861                                   1.00                                       32.93    ATOM        1690            CA SER 425 49.394                            86.494                               189.904                                   1.00                                       30.92    ATOM        1698            CA GLN 426 48.176                            90.276                               189.999                                   1.00                                       29.47    ATOM        1710            CA MET 427 48.379                            91.899                               193.372                                   1.00                                       31.04    ATOM        1719            CA GLU 428 44.978                            93.418                               193.655                                   1.00                                       42.94    ATOM        1729            CA THR 429 45.965                            96.999                               194.423                                   1.00                                       48.52    ATOM        1738            CA SER 430 43.656                            99.720                               193.224                                   1.00                                       48.67    ATOM        1746            CA CYS 431 46.604                           101.354                               191.803                                   1.00                                       52.34    ATOM        1753            CA GLN 432 45.813                           102.789                               188.396                                   1.00                                       50.77    ATOM        1765            CA ILE 433 47.826                           103.166                               185.221                                   1.00                                       51.17    ATOM        1774            CA THR 434 46.923                           105.057                               182.065                                   1.00                                       52.62    ATOM        1783            CA PHE 435 46.641                           103.293                               178.672                                   1.00                                       50.53    ATOM        1795            CA GLU 436 45.167                           103.968                               175.164                                   1.00                                       50.56    ATOM        1805            CA PHE 437 42.643                           101.271                               174.193                                   1.00                                       46.56    ATOM        1817            CA VAL 438 39.404                           101.233                               172.116                                   1.00                                       52.14    ATOM        1825            CA ASP 439 36.342                           103.107                               173.476                                   1.00                                       59.69    ATOM        1834            CA GLN 440 33.750                           100.386                               173.243                                   1.00                                       63.82    ATOM        1846            CA GLU 441 30.764                           102.682                               173.229                                   1.00                                       64.79    ATOM        1856            CA GLN 442 31.810                           104.569                               170.091                                   1.00                                       59.01    ATOM        1868            CA LEU 443 32.994                           101.521                               168.139                                   1.00                                       55.78    ATOM        1877            CA LYS 444 30.279                            99.137                               169.086                                   1.00                                       54.82    ATOM        1890            CA ASP 445 30.862                            96.668                               166.093                                   1.00                                       51.31    ATOM        1899            CA PRO 446 32.144                            93.267                               167.135                                   1.00                                       47.34    ATOM        1906            CA VAL 447 34.363                            92.592                               164.111                                   1.00                                       41.42    ATOM        1914            CA CYS 448 35.680                            96.184                               163.845                                   1.00                                       39.44    ATOM        1921            CA TYR 449 36.273                            96.949                               167.607                                   1.00                                       40.33    ATOM        1935            CA LEU 450 38.224                            93.580                               167.624                                   1.00                                       30.87    ATOM        1944            CA LYS 451 39.989                            94.664                               164.462                                   1.00                                       34.49    ATOM        1957            CA LYS 452 40.962                            97.918                               166.163                                   1.00                                       35.38    ATOM        1970            CA ALA 453 41.503                            96.317                               169.572                                   1.00                                       28.00    ATOM        1976            CA PHE 454 44.012                            93.845                               168.164                                   1.00                                       32.56    ATOM        1988            CA LEU 455 46.236                            96.653                               166.861                                   1.00                                       40.19    ATOM        1997            CA LEU 456 46.402                            98.605                               170.114                                   1.00                                       42.45    ATOM        2006            CA VAL 457 46.966                            95.358                               172.060                                   1.00                                       38.09    ATOM        2014            CA GLN 458 50.298                            95.232                               170.161                                   1.00                                       48.02    ATOM        2026            CA ASP 459 51.630                            98.529                               171.587                                   1.00                                       41.96    ATOM        2035            CA ILE 460 50.230                            97.686                               175.00i                                   1.00                                       25.82    ATOM        2044            CA MET 461 52.409                            94.542                               175.085                                   1.00                                       33.69    ATOM        2053            CA GLU 462 55.702                            96.253                               173.969                                   1.00                                       45.36    ATOM        2063            CA ASP 463 55.069                            99.093                               176.249                                   1.00                                       48.41    ATOM        2072            CA THR 464 52.771                            98.555                               179.186                                   1.00                                       41.63    ATOM        2081            CA MET 465 52.743                            94.854                               180.171                                   1.00                                       42.05    ATOM        2090            CA ARG 466 56.333                            94.928                               181.338                                   1.00                                       44.99    ATOM        2107            CA PHE 467 57.618                            91.923                               183.501                                   1.00                                       38.72    ATOM        2119            CA ARG 468 61.258                            90.894                               184.513                                   1.00                                       50.64    ATOM        2136            CA ASP 469 63.046                            88.868                               181.895                                   1.00                                       53.46    ATOM        2145            CA ASN 470 62.896                            85.096                               181.832                                   1.00                                       52.84    ATOM        2156            CA THR 471 60.211                            84.798                               184.479                                   1.00                                       47.58    ATOM        2165            CA PRO 472 57.055                            82.652                               183.744                                   1.00                                       46.82    ATOM        2172            CA ASN 473 54.971                            85.777                               183.172                                   1.00                                       38.75    ATOM        2183            CA ALA 474 57.431                            87.402                               180.828                                   1.00                                       37.27    ATOM        2189            CA ILE 475 57.795                            84.219                               178.807                                   1.00                                       32.85    ATOM        2198            CA ALA 476 54.021                            84.034                               178.455                                   1.00                                       27.98    ATOM        2204            CA ILE 477 53.844                            87.522                               176.756                                   1.00                                       30.47    ATOM        2213            CA VAL 478 56.690                            86.495                               174.201                                   1.00                                       30.40    ATOM        2221            CA GLN 479 54.381                            83.711                               173.325                                   1.00                                       31.18    ATOM        2233            CA LEU 480 51.419                            86.038                               172.893                                   1.00                                       27.44    ATOM        2242            CA GLN 481 53.392                            88.551                               170.821                                   1.00                                       31.59    ATOM        2254            CA GLU 482 54.572                            85.683                               168.506                                   1.00                                       29.90    ATOM        2264            CA LEU 483 50.900                            84.532                               168.340                                   1.00                                       22.16    ATOM        2273            CA SER 484 50.077                            88.156                               167.451                                   1.00                                       29.37    ATOM        2281            CA LEU 485 52.410                            87.880                               164.490                                   1.00                                       32.70    ATOM        2290            CA ARG 486 50.865                            84.572                               163.229                                   1.00                                       30.59    ATOM        2307            CA LEU 487 47.396                            86.383                               163.343                                   1.00                                       31.02    ATOM        2316            CA LYS 488 48.292                            89.399                               161.352                                   1.00                                       34.90    ATOM        2329            CA SER 489 47.728                            87.397                               158.200                                   1.00                                       35.68    ATOM        2337            CA CYS 490 44.112                            87.134                               159.417                                   1.00                                       35.03    ATOM        2344            CA PHE 491 43.520                            90.786                               159.602                                   1.00                                       42.73    ATOM        2356            CA THR 492 43.145                            93.174                               156.663                                   1.00                                       56.24    ATOM        2365            CA ALA 493 44.877                            96.467                               156.617                                   1.00                                       57.56    ATOM        2371            CA ASP 494 42.318                            99.142                               157.302                                   1.00                                       57.16    ATOM        2380            CA ALA 495 42.063                           102.909                               156.516                                   1.00                                       62.14    ATOM        2386            CA GLU 496 45.542                           104.198                               157.805                                   1.00                                       64.30    ATOM        2396            CA GLU 497 44.589                           107.547                               159.382                                   1.00                                       70.67    ATOM        2406            CA HIS 498 41.577                           105.660                               160.752                                   1.00                                       75.16    ATOM        2419            CA ASP 499 44.400                           103.329                               161.968                                   1.00                                       72.11    ATOM        2428            CA LYS 500 44.773                           105.712                               164.874                                   1.00                                       65.63    ATOM        2441            CA ALA 501 41.040                           106.628                               165.617                                   1.00                                       55.63    ATOM        2447            CA CYS 502 38.563                           105.427                               168.336                                   1.00                                       53.33    ATOM        2454            CA VAL 503 41.155                           105.639                               171.100                                   1.00                                       52.46    ATOM        2462            CA ARG 504 39.866                           106.944                               174.425                                   1.00                                       51.00    ATOM        2479            CA THR 505 42.588                           106.921                               117.117                                   1.00                                       50.42    ATOM        2488            CA PHE 506 41.955                           104.935                               180.331                                   1.00                                       43.64    ATOM        2500            CA TYR 507 43.306                           105.190                               183.809                                   1.00                                       41.84    ATOM        2514            CA GLU 508 42.651                           101.611                               185.218                                   1.00                                       39.35    ATOM        2524            CA THR 509 44.156                            99.096                               187.597                                   1.00                                       41.67    ATOM        2533            CA PRO 510 46.618                            96.563                               185.969                                   1.00                                       39.70    ATOM        2540            CA LEU 511 44.134                            93.600                               186.416                                   1.00                                       42.72    ATOM        2549            CA GLN 512 41.460                            95.632                               184.624                                   1.00                                       37.47    ATOM        2561            CA LEU 513 43.895                            96.148                               181.765                                   1.00                                       27.02    ATOM        2570            CA LEU 514 44.769                            92.421                               181.621                                   1.00                                       24.55    ATOM        2579            CA GLU 515 41.076                            91.327                               181.688                                   1.00                                       31.93    ATOM        2589            CA LYS 516 40.903                            93.442                               178.515                                   1.00                                       31.96    ATOM        2602            CA VAL 517 43.981                            91.949                               176.692                                   1.00                                       26.46    ATOM        2610            CA LYS 518 42.528                            88.538                               177.852                                   1.00                                       30.83    ATOM        2623            CA ASN 519 39.106                            89.397                               176.432                                   1.00                                       37.62    ATOM        2634            CA VAL 520 40.221                            90.488                               172.933                                   1.00                                       35.75    ATOM        2642            CA PHE 521 42.191                            87.109                               172.662                                   1.00                                       25.45    ATOM        2654            CA ASN 522 39.292                            85.079                               173.953                                   1.00                                       26.73    ATOM        2665            CA GLU 523 36.870                            86.722                               171.437                                   1.00                                       33.04    ATOM        2675            CA THR 524 39.260                            86.923                               168.369                                   1.00                                       29.70    ATOM        2684            CA LYS 525 39.423                            83.195                               169.011                                   1.00                                       28.41    ATOM        2697            CA ASN 526 35.624                            82.663                               169.124                                   1.00                                       34.46    ATOM        2708            CA LEU 527 34.816                            84.653                               166.021                                   1.00                                       28.09    ATOM        2717            CA LEU 528 37.617                            82.902                               164.068                                   1.00                                       25.62    ATOM        2726            CA ASP 529 35.899                            79.662                               164.964                                   1.00                                       35.00    ATOM        2735            CA LYS 530 32.659                            81.068                               163.537                                   1.00                                       37.54    ATOM        2748            CA ASP 531 34.250                            82.150                               160.272                                   1.00                                       37.92    ATOM        2757            CA TRP 532 38.053                            82.453                               159.573                                   1.00                                       40.02    ATOM        2773            CA ASN 533 38.395                            85.446                               157.159                                   1.00                                       42.64    ATOM        2784            CA ILE 534 35.840                            87.437                               159.393                                   1.00                                       39.27    ATOM        2793            CA PHE 535 38.635                            89.996                               139.881                                   1.00                                       40.49    ATOM        2805            CA SER 536 38.485                            90.720                               156.238                                   1.00                                       46.56    ATOM        2813            CA LYS 537 35.844                            93.371                               157.102                                   1.00                                       45.61    ATOM        2826            CA ASN 538 35.653                            96.936                               155.760                                   1.00                                       54.39    ATOM        2837            CA CYS 539 35.786                            98.626                               159.004                                   1.00                                       50.55    ATOM        2844            CA ASN 540 36.738                           101.888                               157.286                                   1.00                                       58.49    ATOM        2855            CA ALA 541 33.143                           102.934                               157.648                                   1.00                                       61.90    ATOM        2861            CA SER 542 32.854                           101.855                               161.326                                   1.00                                       56.62    ATOM        2869            CA PHE 543 35.911                           103.888                               162.235                                   1.00                                       55.25    ATOM        2881            CA ALA 544 33.885                           107.102                               161.741                                   1.00                                       61.94    ATOM        2887            CA GLU 545 30.949                           107.003                               164.203                                   1.00                                       63.83    ATOM        2897            CA CYS 546 33.917                           107.418                               166.572                                   1.00                                       61.90    ATOM        2904            CA SER 547 33.751                           110.878                               168.150                                   1.00                                       66.22    ATOM        2912            CA ALA 548 37.461                           110.300                               168.811                                   1.00                                       65.91    END    __________________________________________________________________________

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 10    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 56 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    AGGTGTCTCATAGAAAGTTCGGACGCAGGCCTTGTCATGCTCTTCATAATCCTTGG56    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 58 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    CAGGAGAAAGCTTATGTCTGAATATTGTAGCGCCATGATTGGGAGTGGAGCCCTGCAG58    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 58 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    CAGGAGAAAGCTTATGTCTGAATATTGTAGCGCCATGATTGGGAGTGGACACCTGCAG58    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 58 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    CAGGAGAAAGCTTATGTCTGAATATTGTAGCCACATGATTGGGAGTGGAGCCCTGCAG58    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 72 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    AGGAGAAAGCTTATGTCTGAATATTGTAGCCACATGATTGGGAGTGGACACCTGCAGTCT60    CTGGCTCGGCTG72    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 85 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    GGACGCAGGCCTTGTCATGCTCTTCATAATCCTTGGTGAAGCAGCTCTTCAGCCTCAAAG60    AGAGTTCCTGCAGCTGTTTAATGGC85    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 76 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    CATGACAAGGCCTGCGTCCGAACTTTCTATGAGACACCTCTCCAGTTGCTGGCGAAGGTC60    AAGGCTGTCTTTAATG76    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    GGATCAGGATCCCTCGGACTGCCTCTC27    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    GCGTACCATGGGCCCAGGAGTTCTGC26    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 33 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    AGTCGAGGATCCTCAATCCGGGGGATGCGTGTG33    __________________________________________________________________________

What is claimed is:
 1. Crystalline macrophage colony stimulating factorα (M-CSFα), the M-CSFα being a dimer of two M-CSFα polypeptide monomers,said monomers being the same or different and having the same amino acidsequence as a mature M-CSFα polypeptide, and when said monomers aredifferent, said monomers differing from one another by at least one butless than five amino acid residues.
 2. The crystalline M-CSFα of claim 1having a crystal lattice in the P2₁ 2₁ 2₁ space group.
 3. Thecrystalline M-CSFα of claim 1 wherein the M-CSFα is biologically activewhen resolubilized.
 4. The crystalline M-CSFα of claim 1 wherein the twoM-CSFα polypeptide monomers are disulfide linked to each other.
 5. Thecrystalline M-CSFα of claim 1 wherein each polypeptide monomer has anamino acid sequence between about 145 and 156 amino acids in length, andwherein said sequence has a carboxylic acid terminus located betweenabout 145 and 156 residues measured from the amino acid terminus of saidM-CSFα polypeptide.
 6. The crystalline M-CSFα of claim 1 wherein atleast one of the polypeptide monomers contains residues 4-158 of amature M-CSFα polypeptide.
 7. The crystalline M-CSFα of claim 6 whereinat least one of said polypeptide monomers has at least one but less thanfive amino acid substitutions at a residue or residues selected from thegroup consisting of His 15, Gln 17, Gln 79, Arg 86, Glu 115, Lys 93, Asp99, Leu 55, Ser 18, Gln 20, Arg 21, Ile 75, Val 78, Leu 85, Asp 69, Asn70, His 9, Asn 63 and Thr 34 and said amino acid substitutions are asrecited in Table
 1. 8. The crystalline M-CSFα of claim 5 wherein atleast one of said polypeptide monomers has at least one but less thanfive amino acid substitutions at a residue or residues selected from thegroup consisting of His 15, Gln 17, Gln 79, Arg 86, Glu 115, Lys 93, Asp99, Leu 55, Ser 18, Gln 20, Arg 21, Ile 75, Val 78, Leu 85, Asp 69, Asn70, His 9, Asn 63, and Thr 34 and said amino acid substitutions are asrecited in Table
 1. 9. The crystalline M-CSFα of claim 1 wherein atleast one of the M-CSFα polypeptide monomers is unglycoslyated.
 10. Thecrystalline M-CSFα according to claim 1 wherein at least one of saidpolypeptide monomers has at least one but less than five amino acidsubstitutions at a residue or residues selected from the groupconsisting of His 15, Gln 17, Gln 79, Arg 86, Glu 115, Lys 93, Asp 99,Leu 55, Ser 18, Gln 20, Arg 21, Ile 75, Val 78, Leu 85, Asp 69, Asn 70,His 9, Asn 63, and Thr 34 and said amino acid substitutions are asrecited in Table
 1. 11. A method of crystallizing a macrophage colonystimulating factor α (M-CSFα) dimer having two M-CSFα polypeptidemonomers which may be the same or different, each monomer containing 146to 162 amino acids, when said monomers are different, said monomersdiffering from one another by at least one but less than five amino acidresidues, the method comprising the steps of:a) mixing a solution of theM-CSFα dimer with a precipitant whereby an M-CSFα mixture is formed; b)precipitating crystalline M-CSFα dimer; and c) isolating the crystallineM-CSFα dimer.
 12. The method of claim 11 wherein the precipitantcomprises polyethylene glycol.
 13. The method of claim 11 wherein theM-CSFα dimer is produced from recombinant M-CSFα isolated from abacterial cell.
 14. The method of claim 11 wherein at least one of thepolypeptide monomers is unglycosylated.
 15. The method of claim 11wherein at least one of the polypeptide monomers contains residues 4 to158 of mature M-CSFα polypeptide.
 16. The method of claim 15 wherein atleast one of said polypeptide monomers has at least one but less thanfive amino acid substitutions at a residue or residues selected from thegroup consisting of His 15, Gln 17, Gln 79, Arg 86, Glu 115, Lys 93, Asp99, Leu 55, Ser 18, Gln 20, Arg 21, Ile 75, Val 78, Leu 85, Asp 69, Asn70, His 9, Asn 63, and Thr
 34. 17. The method of claim 11 wherein thecrystalline M-CSFα dimer is biologically active when resolubilized. 18.The method of claim 11 wherein the step of precipitating the M-CSFαcrystal comprises equilibrating the M-CSFα dimer mixture with aprecipitant solution.
 19. The method of claim 18 wherein the precipitantsolution comprises a higher concentration of precipitant than the M-CSFαmixture.
 20. The method of claim 18 wherein the step of equilibratingcomprises applying the M-CSFα mixture to a surface and equilibrating theapplied M-CSFα mixture with a reservoir of the precipitant solution. 21.The method of claim 11 wherein the step of precipitating the crystallineM-CSFα dimer comprises a step of applying M-CSFα seed crystals to theM-CSFα dimer mixture.
 22. The method of claim 11 wherein the step ofprecipitating the M-CSFα dimer comprises the step of altering thetemperature of the M-CSFα mixture.
 23. The method of claim 11 wherein atleast one of said polypeptide monomers has at least one but less thanfive amino acid substitutions at a residue or residues selected from thegroup consisting of His 15, Gln 17, Gln 79, Arg 86, Glu 115, Lys 93, Asp99, Leu 55, Ser 18, Gln 20, Arg 21, Ile 75, Val 78, Leu 85, Asp 69, Asn70, His 9, Asn 63, and Thr
 34. 24. Crystalline macrophage colonystimulating factor α (M-CSFα) comprising amino acids 4-158 of a matureM-CSFα.
 25. The crystalline M-CSF of claim 24 comprising crystals ofM-CSF having measurements of at least about 0.3 mm in at least twodimensions.
 26. The crystalline M-CSF of claim 24 which is capable ofdiffracting X-ray radiation to produce a diffraction patternrepresenting the three-dimensional structure of the M-CSF.
 27. Thecrystalline M-CSF of claim 26 wherein the diffraction pattern representsthe three-dimensional structure of a portion of the M-CSFα (4-158) to anaccuracy of at least five angstroms, wherein the portion includes aminoacid residues between residue 4 and about residue
 153. 28. A method fordetermining a three-dimensional stucture of an M-CSF dimer containingtwo M-CSFα polypeptide monomers that may be the same or different, eachmonomer having between about 146 and 162 amino acids beginning fromabout residue 1 to about residue 5 of mature M-CSFα, when said monomersare different, said monomers differing from one another by at least onebut less than five amino acid residues, the method comprising:a)crystallizing the M-CSF dimer; b) irradiating the crystalline M-CSF toobtain a diffraction pattern characteristic of the crystalline M-CSF;and c) transforming the diffraction pattern into the three-dimensionalstructure of the M-CSF dimer.
 29. A composition comprising:a) a productresulting from the combination of crystalline macrophage colonystimulating factor α (M-CSFα); and b) a carrier compatible with thestability of M-CSFα.
 30. The composition of claim 29 wherein thecrystalline dimer has a crystal lattice in the P2₁ 2₁ 2₁ space group.31. The composition of claim 30 wherein the M-CSFα is biologicallyactive when resolubilized.
 32. The composition of claim 29 wherein thetwo M-CSFα polypeptide monomers are disulfide linked to each other. 33.The composition of claim 29 wherein each monomer has an amino acidsequence between about 145 and about 156 amino acids in length, andwherein said sequence has a carboxylic acid terminus located betweenabout 145 and 156 residues measured from the amino terminus of a M-CSFαpolypeptide.
 34. The composition of claim 33 wherein at least one of thepolypeptides contains residues 4-158 of mature M-CSFα polypeptide. 35.The composition of claim 29 wherein at least one of the M-CSFαpolypeptide monomers is unglycosylated.
 36. The composition of claim 29wherein said M-CSFα has at least one but less than five amino acidsubstitutions at a residue or residues selected from the groupconsisting of His 15, Gln 17, Gln 79, Arg 86, Glu 115, lys 93, Asp 99,Leu 55, Ser 18, Gln 20, Arg 21, Ile 75, Val 78, Leu 85, Asp 69, Asn 70,His 9, Asn 63, and Thr
 34. 37. The composition of claim 33 wherein saidM-CSFα is a dimer of two M-CSFα monomers wherein at least one of saidtwo M-CSFα monomers has the same amino acid sequence as a mature M-CSFα,the second of said two monomers having an amino acid sequence that isthe same or different than the first of said two monomers, wherein whenthe sequences of said monomers are different, said monomers differ by atleast one but less than five amino acid residues.
 38. The composition ofclaim 33 wherein at least one of said M-CSFα monomers has at least onebut less than five amino acid substitutions at a residue or residuesselected from the group consisting of His 15, Gln 17, Gln 79, Arg 86,Glu 115, Lys 93, Asp 99, Leu 55, Ser 18, Gln 20, Arg 21, Ile 75, Val 78,Leu 85, Asp 69, Asn 70, His 9, Asn 63 and Thr 34 and said amino acidsubstitutions are as recited in Table
 1. 39. The composition of claim 34wherein at least one of said polypeptide monomers has at least one butless than five amino acid substitutions at a residue or residuesselected from the group consisting of His 15, Gln 17, Gln 79, Arg 86,Glu 115, Lys 93, Asp 99, Leu 55, Ser 18, Gln 20, Arg 21, Ile 75, Val 78,Leu 85, Asp 69, Asn 70, His 9, Asn 63 and Thr 34 and said amino acidsubstitutions are as recited in Table
 1. 40. A crystalline M-CSFα(4-158) comprising at least one but less than five amino acidsubstitutions at a residue or residues selected from the groupconsisting of His 15, Gln 17, Gln 79, Arg 86, Glu 115, Lys 93, Asp 99,Leu 55, Ser 18, Gln 20, Arg 21, Ile 75, Val 78, Leu 85, Asp 69, Asn 70,His 9, Asn 63 and Thr
 34. 41. A crystalline M-CSFα wherein the M-CSFα isa dimer of two M-CSFα polypeptide monomers.
 42. A crystalline M-CSFwherein the M-CSF is a dimer of two M-CSF polypeptide monomers that arethe same or different, having a length up to residue position 158 asmeasured from the N-terminus of mature M-CSF, and are selected from thegroup consisting of M-CSFα, M-CSFβ and M-CSFγ, when said monomers aredifferent, said monomers differing from one another by at least one butless than five amino acid residues.